U.S. patent application number 17/011981 was filed with the patent office on 2021-07-22 for engineered yeast as a method for bioremediation.
This patent application is currently assigned to Massachusetts Institute of Technology. The applicant listed for this patent is Massachusetts Institute of Technology. Invention is credited to Angela M. Belcher, George Le-Le Sun.
Application Number | 20210221719 17/011981 |
Document ID | / |
Family ID | 1000005495270 |
Filed Date | 2021-07-22 |
United States Patent
Application |
20210221719 |
Kind Code |
A1 |
Sun; George Le-Le ; et
al. |
July 22, 2021 |
ENGINEERED YEAST AS A METHOD FOR BIOREMEDIATION
Abstract
Metal bioremediation and metal mining strategies can include
compositions and methods.
Inventors: |
Sun; George Le-Le; (Culver
City, CA) ; Belcher; Angela M.; (Lexington,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Massachusetts Institute of Technology |
Cambridge |
MA |
US |
|
|
Assignee: |
Massachusetts Institute of
Technology
Cambridge
MA
|
Family ID: |
1000005495270 |
Appl. No.: |
17/011981 |
Filed: |
September 3, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15887305 |
Feb 2, 2018 |
10766798 |
|
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17011981 |
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62453609 |
Feb 2, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/1085 20130101;
C07K 2319/03 20130101; C12N 1/16 20130101; C02F 2101/20 20130101;
C12Y 404/01008 20130101; C12Y 102/01011 20130101; C12Y 203/01031
20130101; C22B 3/24 20130101; C12N 1/063 20130101; C12N 9/88
20130101; C12Y 205/01047 20130101; E21C 41/22 20130101; C02F
2101/203 20130101; E21C 41/32 20130101; C02F 3/347 20130101; C12N
9/1029 20130101; C22B 3/18 20130101; C12N 9/0008 20130101; C02F
3/322 20130101; C07K 14/825 20130101; C02F 2103/10 20130101; Y02P
10/20 20151101; C02F 3/342 20130101 |
International
Class: |
C02F 3/34 20060101
C02F003/34; C02F 3/32 20060101 C02F003/32; E21C 41/22 20060101
E21C041/22; C07K 14/825 20060101 C07K014/825; C12N 1/06 20060101
C12N001/06; C12N 1/16 20060101 C12N001/16; C12N 9/02 20060101
C12N009/02; C12N 9/10 20060101 C12N009/10; C12N 9/88 20060101
C12N009/88; E21C 41/32 20060101 E21C041/32 |
Claims
1-67. (canceled)
68. A composition for remediating a metal to treat water
comprising: a cell expressing a membrane metal transporter, wherein
the membrane metal transporter has specificity for a metal; a
vacuole transporter; and a metal sequestration protein.
69. The composition of claim 68, wherein the cell is yeast.
70. The composition of claim 69, wherein an ubiquitination ligase
is deleted in the yeast.
71. The composition of claim 69, wherein the ubiquitination ligase
is BSD2.
72. The composition of claim 68, wherein the membrane transporter
is SMF1.
73. The composition of claim 68, wherein the vacuole transporter is
CCC1.
74. The composition of claim 68, wherein the metal sequestration
protein is a phytochelatin synthase.
75. The composition of claim 72, wherein SMF1 is mutated to be
sensitive to the metal.
76. The composition of claim 75, wherein the metal is strontium,
lead or mercury.
77. The composition of claim 72, wherein SMF1 is mutated to destroy
primary ubiquitination sites.
78. The composition of claim 68, wherein the membrane transporter
is Sul1 or Sul2.
79. The composition of claim 78, wherein the metal is chromate.
80. The composition of claim 68, wherein the membrane transporter
is CTR1.
81. The composition of claim 80, wherein the metal is copper.
82. The composition of claim 68, wherein the membrane transporter
is ZRT1.
83. The composition of claim 82, wherein the metal is zinc.
84. The composition of claim 68, wherein the membrane transporter
is FRE1.
85. The composition of claim 84, wherein the metal is iron.
86. A method of remediating a metal to treat water comprising:
preparing a composition comprising: a cell expressing a membrane
metal transporter, wherein the membrane metal transporter has
specificity for a metal; a vacuole transporter; and a metal
sequestration protein; and contacting water with the
composition.
87. The method of claim 86, wherein the metal is lithium.
88. The method of claim 86, wherein the metal is a noble metal.
89. The method of claim 86, wherein the noble metal is gold, silver
or platinum.
90. The method of claim 86, wherein the metal is a rare-earth
metal.
91. The method of claim 86, wherein the rare-earth metal is cerium
(Ce), dysprosium (Dy), erbium (Er), europium (Eu), gadolinium (Gd),
holmium (Ho), lanthanum (La), lutetium (Lu), neodymium (Nd),
praseodymium (Pr), promethium (Pm), samarium (Sm), scandium (Sc),
terbium (Tb), thulium (Tm), ytterbium (Yb) or yttrium (Y).
92. A method of mining a metal comprising: preparing a composition
comprising: a cell expressing a membrane metal transporter, wherein
the membrane metal transporter has specificity for a metal; a
vacuole transporter; and a metal sequestration protein; contacting
water with the composition; and lysing the cell to obtain the
metal.
93. A composition for remediating a metal to treat water
comprising: a cell including a knocked-out enzyme required in
sulfate-assimilation pathway, wherein the cell has specificity for
reactions against a metal.
94. The composition of claim 93, wherein the enzyme is HOM2, MET2,
MET17 or CYS4.
95. The composition of claim 93, wherein a surface of the cell is
modified to display a degenerate sequence that is biased towards
cysteine, histidine, glutamic and aspartic acid residues.
96. A method of making a composition for remediating a metal
comprising: deleting an enzyme required in sulfate-assimilation
pathway in a cell, wherein the cell has specificity for reactions
against a metal; and culturing the cell in a medium supplemented
with cysteine and/or methionine.
97. The method of claim 96, further comprising culturing the cell
in the medium buffered to maintain a pH of the media above 4.
98. A method of forming a metal nanoparticle comprising: preparing
a composition comprising a cell including a knocked-out enzyme
required in sulfate-assimilation pathway, wherein the cell has
specificity for reactions against a metal: contacting the
composition with water; and purifying the metal nanoparticle from
the cell.
99. The method of claim 98, wherein the purifying the metal
nanoparticle from the cell comprises enzymatically digesting the
cell wall, pelleting the cellular debris, and collecting the
supernatant.
100. The method of claim 98, wherein the metal nanoparticle is a
metal sulfide.
101. The method of claim 98, wherein the metal is mercury, cadmium,
zinc, lead, sodium, lithium, nickel, iron, copper, cobalt,
manganese, or a lanthanide.
102. The method of claim 98, further comprising culturing the yeast
in a growth medium with cysteine and/or methionine.
103. The method of claim 98, further comprising tuning a size of
the metal nanoparticle by changing a content of cysteine and/or
methionine.
104. The method of claim 98, further comprising tuning a production
rate of the metal nanoparticle by changing a content of cysteine
and/or methionine.
105. The method of claim 98, wherein a surface of the cell is
modified to display a degenerate sequence that is biased towards
cysteine, histidine, glutamic or aspartic acid residues.
106. A method of remediating a metal to treat water comprising:
preparing a cell including a knocked-out enzyme required in
sulfate-assimilation pathway, wherein the cell has specificity
reactions against for a metal; and contacting water with the
composition.
107. A method of mining a metal comprising: preparing a cell
including a knocked-out enzyme required in sulfate-assimilation
pathway, wherein the cell has specificity reactions against for a
metal; and contacting water with the composition.
Description
CLAIM OF PRIORITY
[0001] This application is a divisional of U.S. patent application
Ser. No. 15/887,305, filed Feb. 2, 2018, now U.S. Pat. No.
10,766,798, which claims the benefit of prior U.S. Provisional
Application No. 62/453,609 filed on Feb. 2, 2017, each of which is
incorporated by reference in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
was submitted electronically in ASCII format in parent application
Ser. No. 15/887,305, and is hereby incorporated by reference in its
entirety. Said ASCII copy, created on Apr. 12, 2018, is named
14952_0550_SL.txt and is 10,600 bytes in size.
TECHNICAL FIELD
[0003] This invention relates to metal bioremediation and metal
mining.
BACKGROUND
[0004] Many heavy metals and organic compounds persistently
contaminate public waters due to manufacturing processes,
agricultural waste, and/or from corroding pipes and infrastructure.
Examples such as lead, chromium, and copper leakage can cause long
term physiological damage, and eventual deterioration of the
respiratory organs and skeletal system. See
www.epa.gov/airtoxics/hlthaf/chromium.html, which is incorporated
by reference in its entirety. Chronic exposure, in most cases due
to lack of water sanitation and monitoring, can cause long term
health problems such as cancer and birth-related defects. See
www.epa.gov/airtoxics/hlthef/hapglossaryrev.htm, which is
incorporated by reference in its entirety.
[0005] Similarly, there are a variety of human-made and
artificially derived organic wastes that pervade the public's water
system that both harm the public and damage the environment.
By-products and runoff from industrial sites such as
trichloroethylene (TCE) and other compounds have been shown to
affect the central nervous system, promote heart failure, and even
cause cancer. See Kjellstrand, P., el al., "Irreversible effects of
trichloroethylene exposure on the central nervous system."
Scandinavian journal of work, environment & health (1980):
40-47, Watson, Rebecca E., et al. "Trichloroethylene-contaminated
drinking water and congenital heart defects: a critical analysis of
the literature." Reproductive Toxicology 21.2 (2006): 117-147, and
Wartenberg, Daniel, Daniel Reyner, and Cheryl Siegel Scott.
"Trichloroethylene and cancer: epidemiologic evidence."
Environmental health perspectives 108. Suppl 2 (2000): 161, each of
which is incorporated by reference in its entirety.
[0006] Similarly, there are human-made reservoirs of waste caused
by mining and drilling. For example, the oil sands, mainly centered
in regions of Canada around the Athabasca watershed, have made a
huge environmental impact due to the massive drilling & mining
in the area. Most of the industrial work in the area has released
toxic pollutants such as arsenic, chromium, lead, mixtures of
reactive hydrocarbons, and irremediable tar. See Kelly, Erin N., et
al., "Oil sands development contributes elements toxic at low
concentrations to the Athabasca River and its tributaries."
Proceedings of the National Academy of Sciences 107.37 (2010):
16178-16183, which is incorporated by reference in its
entirety.
SUMMARY
[0007] In one aspect, a composition for remediating a metal to
treat water can include a cell and a first oligomer of a metal
binding protein expressed on a surface of the cell via a linker,
where the linker can be tethered to the first oligomer of the metal
binding protein and to a surface of the cell, the metal binding
protein has specificity for a metal, and the first oligomer of the
metal binding protein expressed on the surface of the cell is
capable of aggregating with a second oligomer of the metal binding
protein in the water upon binding a metal.
[0008] In certain embodiments, the cell can be yeast.
[0009] In certain embodiments, the linker can be a monomer of the
metal binding protein.
[0010] In certain embodiments, the second oligomer of the metal
binding protein can be secreted from the cell.
[0011] In certain embodiments, the metal can be a divalent
metal.
[0012] In certain embodiments, the metal can be a transition
metal.
[0013] In certain embodiments, the metal can be nickel, iron,
copper, cobalt, lead, cadmium or mercury.
[0014] In certain embodiments, the metal binding protein can be
glutamine synthetase.
[0015] In certain embodiments, the metal binding protein can be
fused with a high affinity protein including a metal binding
domain, wherein the metal binding domain has specificity for the
metal.
[0016] In certain embodiments, the high affinity protein can be a
plant metallothionein.
[0017] In certain embodiments, the metal binding protein can be
expressed in a yeast host strain.
[0018] In another aspect, a method of remediating a metal to treat
water can include preparing a composition comprising a cell and a
first oligomer of a metal binding protein expressed on a surface of
the cell via a linker, where the linker can be tethered to the
first oligomer of the metal binding protein and to a surface of the
cell, the metal binding protein has specificity for a metal, and
the first oligomer of the metal binding protein expressed on the
surface of the cell is capable of aggregating with a second
oligomer of the metal binding protein in the water upon binding a
metal, and contacting water with the composition.
[0019] In certain embodiments, the method can further include
adding the second oligomer of the metal binding protein in the
water.
[0020] In certain embodiments, the cell can be yeast.
[0021] In certain embodiments, the linker can be a monomer of the
metal binding protein.
[0022] In certain embodiments, the second oligomer of the metal
binding protein can be secreted from the cell.
[0023] In certain embodiments, the metal can be a divalent
metal
[0024] In certain embodiments, the metal can be nickel, iron,
copper, cobalt, lead, cadmium or mercury.
[0025] In certain embodiments, the metal binding protein can be
glutamine synthetase.
[0026] In certain embodiments, the metal binding protein can be
fused with a high affinity protein including a metal binding
domain, wherein the metal binding domain has specificity for the
metal.
[0027] In certain embodiments, the high affinity protein can be a
plant metallothionein.
[0028] In certain embodiments, the second oligomer of the metal
binding protein can be secreted from the cell via a signal
peptide.
[0029] In certain embodiments, the signal peptide can be S.
cerevisiae's .alpha.-mating-factor.
[0030] In certain embodiments, the signal peptide can be AGA1/2 or
EXG1.
[0031] In certain embodiments, the method can be performed at
20.degree. C. or lower temperature.
[0032] In certain embodiments, the metal binding protein can be
expressed in a yeast host strain.
[0033] In certain embodiments, the yeast host strain can be Pichia
pastoris.
[0034] In another aspect, a method of making a composition for
remediating a metal to treat water can include selecting a cell,
and expressing a metal binding protein on a surface of the cell,
and tethering a first oligomer of a metal binding protein expressed
on a surface of the cell via a linker, where the linker is tethered
to the first oligomer of the metal binding protein and to a surface
of the cell, the metal binding protein has specificity for a metal,
and the first oligomer of the metal binding protein expressed on
the surface of the cell is capable of aggregating with a second
oligomer of the metal binding protein in the water upon binding a
metal.
[0035] In certain embodiments, the cell can be yeast.
[0036] In certain embodiments, the linker can be a monomer of the
metal binding protein.
[0037] In certain embodiments, the second oligomer of the metal
binding protein can be secreted from the cell.
[0038] In certain embodiments, the metal can be a divalent
metal.
[0039] In certain embodiments, the metal can be a transition
metal.
[0040] In certain embodiments, the metal can be nickel, iron,
copper, cobalt, lead, cadmium or mercury.
[0041] In certain embodiments, the metal binding protein can be
glutamine synthetase.
[0042] In certain embodiments, the method can further include
appending a high affinity protein including to the metal binding
protein, where the high affinity protein including a metal binding
domain, wherein the metal binding domain has specificity for the
metal.
[0043] In certain embodiments, the high affinity protein can be a
plant metallothionein.
[0044] In certain embodiments, the method can further include
expressing the metal binding protein in a yeast host strain.
[0045] In another aspect, a method of mining a metal can include
preparing a composition including a cell and a first oligomer of a
metal binding protein expressed on a surface of the cell via a
linker, where the linker can be tethered to the first oligomer of
the metal binding protein and to a surface of the cell, the metal
binding protein has specificity for a metal and the first oligomer
of the metal binding protein expressed on the surface of the cell
is capable of aggregating with a second oligomer of the metal
binding protein in the water upon binding a metal, contacting water
with the composition and lysing the cell to obtain the metal.
[0046] In certain embodiments, the cell can be yeast.
[0047] In certain embodiments, the linker can be a monomer of the
metal binding protein.
[0048] In certain embodiments, the second oligomer of the metal
binding protein can be secreted from the cell.
[0049] In certain embodiments, the metal can be a divalent
metal.
[0050] In certain embodiments, the metal can be a transition
metal.
[0051] In certain embodiments, the transition metal can be nickel,
iron, copper, cobalt, lead, cadmium or mercury.
[0052] In certain embodiments, the metal can be a noble metal.
[0053] In certain embodiments, the noble metal can be gold, silver
or platinum.
[0054] In certain embodiments, the metal can be a rare-earth
metal.
[0055] In certain embodiments, the rare-earth metal can be cerium
(Ce), dysprosium (Dy), erbium (Er), europium (Eu), gadolinium (Gd),
holmium (Ho), lanthanum (La), lutetium (Lu), neodymium (Nd),
praseodymium (Pr), promethium (Pm), samarium (Sm), scandium (Sc),
terbium (Tb), thulium (Tm), ytterbium (Yb) or yttrium (Y).
[0056] In certain embodiments, the metal binding protein can be
glutamine synthetase.
[0057] In certain embodiments, the metal binding protein can be
fused with a high affinity protein including a metal binding
domain, wherein the metal binding domain has specificity for the
metal.
[0058] In certain embodiments, the high affinity protein can be a
plant metallothionein.
[0059] In certain embodiments, the metal binding protein can be
expressed in a yeast host strain.
[0060] In another aspect, a composition for remediating a metal to
treat water can include a cell expressing a membrane metal
transporter, wherein the membrane metal transporter has specificity
for a metal, a vacuole transporter and a metal sequestration
protein.
[0061] In certain embodiments, the cell can be yeast.
[0062] In certain embodiments, an ubiquitination ligase can be
deleted in the yeast.
[0063] In certain embodiments, the ubiquitination ligase can be
BSD2.
[0064] In certain embodiments, the membrane transporter can be SMF
1.
[0065] In certain embodiments, the vacuole transporter can be
CCC1.
[0066] In certain embodiments, the metal sequestration protein can
be a phytochelatin synthase.
[0067] In certain embodiments, the metal can be a divalent
metal.
[0068] In certain embodiments, the metal can be a transition
metal.
[0069] In certain embodiments, the metal can be cadmium.
[0070] In certain embodiments, SMF1 can be mutated to be sensitive
to the metal.
[0071] In certain embodiments, the metal can be strontium, lead or
mercury.
[0072] In certain embodiments, SMF1 can be mutated to destroy
primary ubiquitination sites.
[0073] In certain embodiments, the membrane transporter can be Sull
or Sul2.
[0074] In certain embodiments, the metal can be chromate.
[0075] In certain embodiments, the membrane transporter can be
CTR1.
[0076] In certain embodiments, the metal can be copper.
[0077] In certain embodiments, the membrane transporter can be
ZRT1.
[0078] In certain embodiments, the metal can be zinc.
[0079] In certain embodiments, the membrane transporter can be
FRE1.
[0080] In certain embodiments, the metal can be iron.
[0081] In another aspect, a method of remediating a metal to treat
water can include preparing a composition include a cell expressing
a membrane metal transporter, where the membrane metal transporter
has specificity for a metal, a vacuole transporter, and a metal
sequestration protein, and contacting water with the
composition.
[0082] In certain embodiments, the cell can be yeast.
[0083] In certain embodiments, an ubiquitination ligase can be
deleted in the yeast.
[0084] In certain embodiments, the ubiquitination ligase can be
BSD2.
[0085] In certain embodiments, the membrane transporter can be
SMF1.
[0086] In certain embodiments, the vacuole transporter can be
CCC1.
[0087] In certain embodiments, the metal sequestration protein can
be a phytochelatin synthase.
[0088] In certain embodiments, the metal can be a divalent
metal.
[0089] In certain embodiments, the metal can be a transition
metal.
[0090] In certain embodiments, the metal can be cadmium.
[0091] In certain embodiments, SMF1 can be mutated to be sensitive
to the metal.
[0092] In certain embodiments, the metal can be strontium, lead or
mercury.
[0093] In certain embodiments, SMF1 can be mutated to destroy
primary ubiquitination sites.
[0094] In certain embodiments, the membrane transporter can be Sul1
or Sul2.
[0095] In certain embodiments, the metal can be chromate.
[0096] In certain embodiments, the membrane transporter can be
CTR1.
[0097] In certain embodiments, the metal can be copper.
[0098] In certain embodiments, the membrane transporter can be
ZRT1.
[0099] In certain embodiments, the metal can be zinc.
[0100] In certain embodiments, the membrane transporter can be
FRE1.
[0101] In certain embodiments, the metal can be iron.
[0102] In another aspect, a method of mining a metal can include
preparing a composition including a cell expressing a membrane
metal transporter, where the membrane metal transporter has
specificity for a metal, a vacuole transporter, and a metal
sequestration protein, contacting water with the composition, and
lysing the cell to obtain the metal.
[0103] In certain embodiments, the cell can be yeast.
[0104] In certain embodiments, an ubiquitination ligase can be
deleted in the yeast.
[0105] In certain embodiments, the ubiquitination ligase can be
BSD2.
[0106] In certain embodiments, the membrane transporter is
SMF1.
[0107] In certain embodiments, the vacuole transporter can be
CCC1.
[0108] In certain embodiments, the metal sequestration protein can
be a phytochelatin synthase.
[0109] In certain embodiments, the metal can be a divalent
metal.
[0110] In certain embodiments, the metal can be a transition
metal.
[0111] In certain embodiments, the transition metal can be nickel,
iron, copper, cobalt, lead, cadmium or mercury.
[0112] In certain embodiments, the metal can be lithium.
[0113] In certain embodiments, the metal can be a noble metal.
[0114] In certain embodiments, the noble metal can be gold, silver
or platinum.
[0115] In certain embodiments, the metal can be a rare-earth
metal.
[0116] In certain embodiments, the rare-earth metal can be cerium
(Ce), dysprosium (Dy), erbium (Er), europium (Eu), gadolinium (Gd),
holmium (Ho), lanthanum (La), lutetium (Lu), neodymium (Nd),
praseodymium (Pr), promethium (Pm), samarium (Sm), scandium (Sc),
terbium (Tb), thulium (Tm), ytterbium (Yb) or yttrium (Y).
[0117] In another aspect, a composition for remediating a metal to
treat water can include a cell including a knocked-out enzyme
required in sulfate-assimilation pathway, where the cell has
specificity for reactions against a metal.
[0118] In certain embodiments, the enzyme can be HOM2, MET2, MET17
or CYS4.
[0119] In certain embodiments, in the metal can be a divalent
metal.
[0120] In certain embodiments, the metal can be a transition
metal.
[0121] In certain embodiments, the metal can be mercury, zinc,
copper, cadmium or lead.
[0122] In certain embodiments, a surface of the cell can be
modified to display a biomineralization peptide.
[0123] In certain embodiments, a surface of the cell can be
modified to display a degenerate sequence that is biased towards
cysteine, histidine, glutamic and aspartic acid residues.
[0124] In another aspect, a method of making a composition for
remediating a metal can include deleting an enzyme required in
sulfate-assimilation pathway in a cell, wherein the cell has
specificity for reactions against a metal and culturing the cell in
a medium supplemented with cysteine and/or methionine.
[0125] In certain embodiments, the enzyme can be HOM2, MET2, MET17
or CYS4.
[0126] In certain embodiments, the metal can be a divalent
metal.
[0127] In certain embodiments, the metal can be a transition
metal.
[0128] In certain embodiments, the metal can be mercury, zinc,
copper, cadmium or lead.
[0129] In certain embodiments, the method can further include
modifying a surface of the cell to display a biomineralization
peptide.
[0130] In certain embodiments, the method can further include
modifying a surface of the cell to display a degenerate sequence
that are biased towards cysteine, histidine, glutamic or aspartic
acid residues.
[0131] In certain embodiments, the method can further include
culturing the cell in the medium supplemented with sodium
sulfide.
[0132] In certain embodiments, the method can further include
culturing the cell in the medium buffered to maintain a pH of the
media above 4.
[0133] In another aspect, a method of forming a metal nanoparticle
can include preparing a composition comprising a cell including a
knocked-out enzyme required in sulfate-assimilation pathway,
wherein the cell has specificity for reactions against a metal,
contacting the composition with water, and purifying the metal
nanoparticle from the cell.
[0134] In certain embodiments, the purifying the metal nanoparticle
from the cell can include enzymatically digesting the cell wall,
pelleting the cellular debris, and collecting the supernatant.
[0135] In certain embodiments, the metal nanoparticle can be HgS,
CdS, ZnS or PbS.
[0136] In certain embodiments, the cell can be yeast.
[0137] In certain embodiments, the method can further include
culturing the yeast in a growth medium with cysteine and/or
methionine.
[0138] In certain embodiments, the method can further include
tuning a size of the metal nanoparticle by changing a content of
cysteine and/or methionine.
[0139] In certain embodiments, the method can further include
tuning a production rate of the metal nanoparticle by changing a
content of cysteine and/or methionine.
[0140] In another aspect, a method of remediating a metal to treat
water can include preparing a cell including a knocked-out enzyme
required in sulfate-assimilation pathway, where the cell has
specificity reactions against for a metal and contacting water with
the composition.
[0141] In certain embodiments, the enzyme can be HOM2, MET2, MET17
or CYS4.
[0142] In certain embodiments, the metal can be copper, cadmium or
lead
[0143] In certain embodiments, a surface of the cell can be
modified to display a biomineralization peptide.
[0144] In certain embodiments, a surface of the cell can be
modified to display a degenerate sequence that is biased towards
cysteine, histidine, glutamic or aspartic acid residues.
[0145] In another aspect, a method of mining a metal can include
preparing a cell including a knocked-out enzyme required in
sulfate-assimilation pathway, where the cell has specificity
reactions against for a metal and contacting water with the
composition.
[0146] In certain embodiments, the enzyme can be HOM2, MET2, MET17
or CYS4.
[0147] In certain embodiments, the metal can be a divalent
metal.
[0148] In certain embodiments, the metal can be a transition
metal.
[0149] In certain embodiments, the transition metal can be mercury,
zinc, copper, cobalt, cadmium or lead.
[0150] In certain embodiments, the metal can be a noble metal.
[0151] In certain embodiments, the noble metal can be gold, silver
or platinum.
[0152] In certain embodiments, the metal can be a rare-earth
metal.
[0153] In certain embodiments, the rare-earth metal can be cerium
(Ce), dysprosium (Dy), erbium (Er), europium (Eu), gadolinium (Gd),
holmium (Ho), lanthanum (La), lutetium (Lu), neodymium (Nd),
praseodymium (Pr), promethium (Pm), samarium (Sm), scandium (Sc),
terbium (Tb), thulium (Tm), ytterbium (Yb) or yttrium (Y).
[0154] In certain embodiments, a surface of the cell can be
modified to display a biomineralization peptide.
[0155] In certain embodiments, a surface of the cell can be
modified to display a degenerate sequence that is biased towards
cysteine, histidine, glutamic or aspartic acid residues.
[0156] Other aspects, embodiments, and features will be apparent
from the following description, the drawings, and the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0157] FIG. 1 shows analogies between physicochemical and
engineered biological processes.
[0158] FIG. 2 shows yeast displayed+multiplier protein system.
[0159] FIGS. 3A-3C show structure of multiplier proteins (GS). FIG.
3A shows crystal structure and TEM images of GS. FIG. 3B shows
controlled protein aggregation of multiplier proteins (GS
monomers). FIG. 3C shows controlled protein aggregation of
multiplier proteins (GS oligomers).
[0160] FIG. 4 shows SEM image of yeast with aggregated GS.
[0161] FIG. 5 shows metal uptake with yeast+GS.
[0162] FIG. 6 shows quantifying GS expression level using FACS.
[0163] FIG. 7 shows staining of membrane transporter SMF1, and
vacuole transporter CCC1.
[0164] FIG. 8 shows effects on engineering yeast transporters on
cadmium uptake.
[0165] FIG. 9 shows metal transport screening pipeline.
[0166] FIG. 10A shows a diagram illustrating plant phytochelatin
TaPCS1 to enhance metal tolerance in yeast. FIG. 10B shows TaPCS1
enhances heavy metal tolerance.
[0167] FIG. 11 shows growth curve of engineered strains in 100
.mu.M cadmium.
[0168] FIGS. 12A-12B show using sulfate permeases to uptake
structurally similar chromate ions. FIG. 12A shows basic electron
density mapping of sulfate and chromate show a high degree of
structural resemblance. FIG. 12B shows overexpressing sulfate
permeases Sul1 and 2 lead to increase chromate uptake.
[0169] FIG. 13A shows simplified diagram of yeast's sulfur pathway.
FIG. 13B shows crystalline metal sulfide particles (e.g. PbS) using
.DELTA.CYS4 strain. FIG. 13C shows crystalline metal sulfide
particles (e.g. PbS) using .DELTA.HOM2. FIG. 13D shows crystalline
metal sulfide particles (e.g. PbS) using .DELTA.MET17. FIG. 13E
shows a deeper look of the particles. FIG. 13F shows
EDX--ratiometric analysis of the metal sulfide particles. FIG. 13G
shows XRD--material crystallinity of the metal sulfide
particles.
[0170] FIG. 14 shows lead acetate strips (left) turn black in the
presence of gaseous sulfur.
[0171] FIGS. 15A-15C show the effects of media condition on sulfide
production. FIG. 15A shows the absence or presence of cysteine or
methionine either increases (.DELTA.CYS4) or decreases sulfide
production (.DELTA.HOM2, MET2, MET17). FIG. 15B shows the absence
of cysteine both reduces the onset of sulfide production and
increases sulfide production yield for .DELTA.MET17. FIG. 15C shows
effects on nutrient conditions on sulfide production.
[0172] FIGS. 16A-16C show precipitation of metal sulfides using
sulfur-producing yeast. FIG. 16A shows cultures grown overnight
with Cu, Cd, Zn, Pb, Hg produce metal sulfides which give
observable color changes indicative of CuS, CdS, ZnS, PbS and HgS
precipitation. FIG. 16B shows analysis under TEM shows that metal
sulfides such as CdS precipitate on the cell wall. FIG. 16C shows
photos of Cds and PbS nanoparticle synthesis with/without
yeast.
[0173] FIG. 17 shows hypothesized schematic of peptide mediated
metal sulfide mineralization.
[0174] FIG. 18 shows proof of concept screening of cadmium and
copper metal sulfide formation using yeast display peptide
libraries (left image). The amount of precipitated CdS can be
modulated by displaying different peptides on the yeast surface.
(right image) Likewise, colonies can be discriminated by colony
color density.
[0175] FIG. 19 shows solubility curves of metals at various pH
values.
[0176] FIGS. 20A-20C shows examples of combining strategies 1-3.
FIG. 20A shows biomineralization can be initiated with released
sulfur compounds that subsequently nucleate on displayed
biomineralization peptides. FIG. 20B shows high valent metals are
reduced to divalent forms which are recognized by divalent metal
transporters such as ZIPs, CTRs, FTRs, etc. FIG. 20C shows
transported metals compartmentalized by a variety of mechanisms
such as phytochelation, mineralization, or vacuole
compartmentalization.
[0177] FIG. 21 shows sequence map of DNA cassette containing AGA1,
AGA2, protein of interest (POI), and TRP1 marker. Annotations
specify relevant sequences such as promoters, terminators, tags,
etc. FIG. 21 discloses SEQ ID NO: 9.
[0178] FIG. 22A shows sequences encoding MTs (or other proteins)
can be rationally designed or mutated to generate a library of
mutants to be tested for improved metal uptake, waste removal, or
waste conversion. FIG. 22B shows libraries screened via yeast
display for metal uptake, or alternatively tested against waste
removal such as TCE. FIG. 22C shows selection based on optical,
colorimetric, or chemical assays to screen for the most efficacious
binders or catalyst via high-throughput platforms such as flow
cytometry or automated plate readers.
[0179] FIG. 23A shows sequence map of GAL1 inducible metal
transporters followed by a V5 epitope tang and a CYC1 transcription
terminator. FIG. 23B shows illustration representing metal uptake
via yeast metal transporters.
[0180] FIG. 24A shows that genes of yeast metal transporters for
zinc (ZRTs), copper (CTRs), iron (FRE), etc. were over-expressed
and expressions were qualitatively assessed using
immunofluorescence by staining the V5 epitope tag. FIG. 24B shows
uptake studies of over-expressed metal transporters for Cu(II)
(blue), Zn(II) (red) and Mn(II) (purple). Highlighted bars indicate
significant increase in uptake compared to WT (dashed line).
[0181] FIG. 25 shows flow diagram of using and recycling DIY yeast
remediation packets for heavy metal removal.
[0182] FIG. 26 shows a simple genetic switch turns on the
production of the green fluorescent protein (GFP) to indicate the
presence of metal contaminants still present in the filtered
drinking water.
[0183] FIGS. 27A-27C show yeast display of plant metallothioneins
(MTs) to demonstrate copper uptake and enhanced survivability in
copper solutions. FIG. 27A shows four families of plant MT proteins
were taken from Arabidopsis Thaliana and expressed in the AGA1 and
AGA2 yeast display system. All families of MTs showed increased
uptake of copper (FIG. 27B) and increased survivability in copper
solutions (FIG. 27C).
[0184] FIGS. 28A-28B show using Percoll density gradients to
fractionate clones with improved metal uptake efficiency (see also
FIG. 9). FIG. 28A shows representative image of the mutant strain
cell band that is higher in density (lower in the gradient) than
compared to wild-type. FIG. 28B shows elemental analysis on
collected cells from FIG. 28A showing a relative increase in metal
uptake.
[0185] FIGS. 29A-29D show engineering metal transporters to enhance
heavy metal uptake. FIG. 29A shows schematic of heavy metal uptake
pathways in yeast. FIG. 29B shows micrographs demonstrating that
metal transporter SMF1 can be engineered to improve cadmium uptake.
FIG. 29C shows cadmium uptake of WT yeast and engineered yeast
strains in .mu.M (top) and mg Cd.sup.2+/g dry weight (DW, bottom).
FIG. 29D shows SMF1 mutagenesis results.
[0186] FIG. 30 shows wine yeast engineered to limit hydrogen
sulfide production.
[0187] FIGS. 31A-31D show engineering yeast strains and detecting
hydrogen sulfide production. FIG. 31A shows lead acetate strips
giving a binary measurement on whether strains are capable of
overproducing sulfur. FIG. 31B shows hydrogen sulfide columns
marked with scales can determine hydrogen sulfide produced in
cultures. FIGS. 31C and 31D show 1 ppm resolution columns (yellow)
undergo a purple change in the presence of hydrogen sulfide,
whereas 5 ppm resolution columns (white) undergo a brown
change.
[0188] FIGS. 32A-32B show that yeast can generate sulfur to
precipitate metals out of solution.
[0189] FIG. 32A cadmium removal using yeast sulfur production
eliminates 90.+-.5% of cadmium in solution. FIG. 32B Precipitated
CdS particles excite at 350 nm and emit at 415 nm which is
characteristic of CdS quantum dot behavior.
[0190] FIGS. 33A-33B show cross section TEM images of yeast cells
with CdS embedded in the cell wall. FIG. 33A shows images showing
speckles of cadmium sulfide on the yeast cell wall. FIG. 33B shows
particles are approximately 10-20 nm in diameter, with some encased
in a biological shell which might be a consequence of cell wall
encasing or during cell wall extraction.
[0191] FIGS. 34A-34B show established infrastructure to produce
water-remediating yeast cultures for global applications. FIG. 34A
shows the engineered yeast can be stored as freeze-dried or
compressed packages, as they normally are in for consumer purposes,
for long-term storage and portability. FIG. 34B shows the beer and
wine industry have already developed a cheap and scalable source of
yeast to scale the production of engineered strains for water
remediation.
DETAILED DESCRIPTION
[0192] Many of the current industrial methods used for water
treatment lack metal specificity and produce significant amounts of
secondary waste which has made waste treatment an unsustainable
process. See Gokhan Ekrem Ustun, Seval Kutlu Akal Solmaz, and As
kin Birgul. Regeneration of industrial district wastewater using a
combination of Fenton process and ion exchange--A case study.
Resources, Conservation and Recycling, 52 (2):425-440, 2007, and M
A Barakat. New trends in removing heavy metals from industrial
wastewater. Arabian Journal of Chemistry, 4 (4):361-377, 2011, each
of which is incorporated by reference in its entirety. The commonly
used methods, ion-exchange, absorption, and chemical precipitation,
also known as physicochemical methods, also have a high cost
barrier preventing easy adoption in developing areas which are more
likely to require intensive heavy metal treatment. See R K Rattan,
S P Datta, P K Chhonkar, K Suribabu, and A K Singh. Long-term
impact of irrigation with sewage effluents on heavy metal content
in soils, crops and groundwater case study. Agriculture, Ecosystems
& Environment, 109 (3):310-322, 2005, which is incorporated by
reference in its entirety.
[0193] Therefore, there needs to be more sustainable technologies
for water treatment that require methods beyond just physical and
chemical techniques. With current bioengineering techniques this
balance between consumption and waste of metals can be exploited to
favor metal accumulation and conversion without causing toxic
effects. See Robert Wysocki and Markus J Tamas. How Saccharomyces
cerevisiae copes with toxic metals and metalloids. FEMS
microbiology reviews, 34 (6):925-951, 2010, which is incorporated
by reference in its entirety. Simple organisms such as yeast can be
genetically modified to act as living agents that sequester and
remove waste from the environment. The added benefit is that yeast
can be easily modified and self-propagate with minimal user
intervention, making them a desirable engineerable platform which
is cheap, scalable, and easily handled.
[0194] Current approaches to clean contaminated waters and
landmasses are to synthesize chelating or reactive molecules to
sequester heavy metals and toxic compounds. See Deshpande,
Kiranmayi, et al., "Efficient sequestration and reduction of of
hexavalent chromium with organosilica sol-gels," Journal of
Materials Chemistry 15.29 (2005): 2997-3004, and Tang, Hao, et al.,
"Reductive dechlorination of activated carbon-adsorbed
trichloroethylene by zero-valent iron: carbon as electron shuttle,"
Journal of environmental quality 40.6 (2011): 1878-1885, each of
which is incorporated by reference in its entirety. However, these
methods are costly and complex in design and are themselves prone
to forming waste by-products. An alternative method can avoid these
problems by using biological peptide chemistry and protein
engineering a facile, modular, and scalable platform that uses
protein binding domains as waste-containment agents. An advantage
over chemically synthesizing polymers or fabricating devices is the
simplicity of designing novel proteins using current gene editing
technology, modularity (whereas chemicals and devices have to redo
the cycle of theorization, construction and validation), and
cost-efficient scalability when considering the large volumes of
contaminated waters to treat. See Needels, Michael C., et al.,
"Generation and screening of an oligonucleotide-encoded synthetic
peptide library," Proceedings of the National Academy of Sciences
90.22 (1993): 10700-10704, and Houghten, Richard A., et al.,
"Generation and use of synthetic peptide combinatorial libraries
for basic research and drug discovery," (1991): 84-86, each of
which is incorporated by reference in its entirety.
[0195] On the other end of the spectrum, current research is
pursuing bioremediation techniques that focus on using natural
organisms to remove or consume waste, thereby providing an
environmentally friendlier and natural alternative for managing
waste, Historically, bioremediation was first invented by George
Robinson in the 1960s by first demonstrating the use of bacteria to
degrade petroleum and other hydrocarbon based pollutants. See
Golding, Lynnea, "Bioremediation of Pesticides," which is
incorporated by reference in its entirety. However, the process in
which organisms break down hazardous substances into less toxic or
usable forms has been a universal concept in all living organisms.
Especially so, the management of metal concentration and
localization in cells plays an incredibly important role in
cellular homeostasis. Of all the species that have adapted to
handle highly polluted environments, the most highly studied are
plants, fungi, and bacteria. See Juwarkar, Asha A., Sanjeev K.
Singh, and Ackmez Mudhoo, "A comprehensive overview of elements in
bioremediation," Reviews in Environmental Science and
Bio/Technology 9.3 (2010): 215-288, and Ron, Eliora Z. and Eugene
Rosenberg, "Biosurfactants and oil bioremediation," Current opinion
in biotechnology 13.3 (2002): 249-252, each of which is
incorporated by reference in its entirety. However, given the
diversity of plants and bacteria and their respective genes and
proteins that play a role in heavy metal uptake bring into question
whether using native organisms with specific environmental and
growth conditions is a foreseeable platform for a creating a
scalable and accessible bioremedation technology. Therefore, the
next step should be in favor of using an engineerable host which
can be manipulated to function analogously to plants and/ or
bacteria by expressing the relevant proteins using current
technologies in molecular and genetic engineering.
[0196] Therefore, protein engineering can be applied on yeast to
create the next biological platform for bioremediation. Yeast have
been a model organism for genetic studies since the age of Louis
Pasteur, and since then yeast are becoming increasingly relevant in
gene expression and protein studies as genetic engineering
technology continues to develop. The field of yeast biology has
already optimized genetic manipulation techniques such as
transformations, genomic recombination, heterologous protein
expression, and design and function of genetic circuits. In
addition, many of the studies that have identified phytochelatins,
metallothioneins, metal transporters, and cytochromes were either
discovered in yeast: or functionally identified using functional
complementation of yeast mutants proving that yeast already contain
the basic machinery for metal transport, uptake, and sequestration.
Expressing the relevant proteins and enzymes can enable uptake and
sequester metals beyond the normal limitations of wild-type yeast
and vastly beyond the hazardous standards set by the EPA.
[0197] Disclosed herein is a method for remediating a metal to
treat water or for mining a metal to take the principles from
ion-exchange, adsorption, and chemical precipitation and create
analogous strategies in yeast. The act of binding metals
(ion-exchange), internalization (adsorption), and conversion
(chemical precipitation) are naturally found in biological
processes for cellular homeostasis. See Julian C Rutherford and
Amanda J Bird. Metal-responsive transcription factors that regulate
iron, zinc, and copper homeostasis in eukaryotic cells. Eukaryotic
cell, 3 (1):1-13, 2004, which is incorporated by reference in its
entirety. In certain embodiments, genetically engineered yeast can
be used as a means to bioremediate waste waters. In certain
embodiments, a combination of yeast display to bind metals onto the
surface of yeast, over-expression of metal transporters to uptake
metals intracellularly, and biochemical pathways that enables to
recycle captured metals using metabolic enzymes can be used. In
parallel, yeast enzymes can be developed to consume and/ or degrade
harmful organic compounds such as TCE generated from mining and
other environmentally unfriendly practices. Given current
bioengineering techniques, these mechanisms can be perturbed to
favor metal accumulation by exposing more metal binding proteins,
increasing activity of metal transporters, or promoting specific
metal reaction pathways, for example. This leads to the creation of
three unique strategies: cell surface display, cell uptake, and
biomineralization (FIG. 1): 1) cell surface display of metal
binding proteins mimics the mechanism of action of surface
functionalized ion-exchange resins; 2) physical uptake of metals
can be achieved by engineering hyperactive metal transporters; and
3) yeast can supply reactive by-products to mineralize metals from
solution, instead of relying on external chemicals for chemical
precipitation. Despite differing approaches, all three can be
optimized based on metal capture capacity, metal specificity and
selectivity, and yeast metal tolerance and survivability. In
certain embodiments, the metal can be a divalent metal. In certain
other embodiments, the metal can be a transition metal.
[0198] These strategies closely mimic the mechanism of action of
the physicochemical methods mentioned above, yet address the
limitations of cost, development time, and scalability. It is
possible to optimize these strategies such that organisms, like
yeast, can be used to treat and manage waste for the environment
and the public.
Yeast Display Capture Capacity
[0199] Yeast display as a method for material capture is limited by
cell density and the expression number of peptide/proteins per cell
surface. Nominal capture capacity is in the micromolar range
assuming culture densities from ten to one hundred thousand cells
per milliliter, expression levels ranging from ten to one hundred
thousand (experimentally determined), and effective binding sites
per peptide/protein between 1 and 10. Uptake can then be defined
as:
uptake[M]=N.sub.c.times.N.sub.e.times.n/N.sub.A
Where
[0200] N.sub.c=density of cells [0201] N.sub.e=expression number
per cell [0202] n=binding sites per expression [0203]
N.sub.A=Avogadro's number (6.022e23)
Density Changes Due to Metal Uptake
[0204] Assuming transporter metal uptake of 100 .mu.M (denoted as
M; empirically determined), and nominal values for yeast volume and
density, then density changes due to metal uptake can be determined
with:
.DELTA..rho. = .rho. + m metals V ##EQU00001##
[0205] Assuming volume does not increase dramatically with metal
internalization. The added mass contributed by the metals can be
calculated with
m.sub.metais=M.times.V.times.MW/N.sub.A
Where
[0206] .rho.=yeast density (1.129 g/mL) [0207] V=yeast volume
(35.times.10.sup.-15 mL) [0208] m.sub.metals=added mass due to
metal [0209] MW=metal molecular weight [0210] N.sub.A=Avogadro's
number (6.022e23)
[0211] Using a lower end molecular weight (MW) of 54.9 (manganese)
and a higher end molecular weight of 207.2 (lead) density changes
can range from 2-25%.
Strategy 1--Cell Surface Capture--Increasing Capture Capacity of
Yeast Display Using "Multiplier" Proteins
[0212] Strategy 1 is focused on overcoming the low metal capture
capacity of yeast display when compared to physicochemical
techniques such as ion-exchange.
[0213] A composition and a method for remediating a metal to treat
water or for mining a metal can include a cell and a first oligomer
of a metal binding protein expressed on a surface of the cell via a
linker, where the linker is tethered to the first oligomer of the
metal binding protein and to a surface of the cell, the metal
binding protein has specificity for a metal; and the first oligomer
of the metal binding protein expressed on the surface of the cell
is capable of aggregating with a second oligomer of the metal
binding protein in the water upon binding a metal. The oligomer can
be an oligonucleotide having 1 to 30 nucleotides, for example, 3 to
25 nucleotides.
[0214] In certain embodiments, a "multiplier" protein system can be
used, where protein monomers tethered to a metal binding protein
(MBP) aggregate onto the yeast surface, effectively multiplying the
number of MBPs displayed per cell. These yeast aggregates are able
to capture 1-10 mM of copper, cobalt, and cadmium at 1 OD.sub.600
of cells; 2-orders of magnitude greater than any existing
techniques on yeast display capture of heavy metals. In certain
embodiments, metal specificity can be controlled by engineering the
tethered MBPs. In certain embodiments, engineered plant
metallothioneins can be used. Engineered plant metallothioneins are
small proteins with metal specificity towards mercury, cadmium,
lead, and a range of other divalent metals. In certain embodiments,
the multiplier protein can be engineered to aggregate in response
to various stimuli, or to be reversible, such that this strategy
can both capture, and then release the collected metal
contaminates.
[0215] In certain embodiments, the metal can be a divalent metal.
In certain other embodiments, the metal can be a transition
metal.
Strategy 2--Metal Uptake--Creating Yeast Hyper Accumulators Using
Membrane and Vacuole Transporters
[0216] Strategy 2 exploits the yeast metal transport system to
hyper accumulate metals present in the environment.
[0217] A composition and a method for remediating a metal to treat
water or for mining a metal can include a cell expressing a
membrane metal transporter, where the membrane metal transporter
has specificity for a metal, a vacuole transporter, and a metal
sequestration protein. The composition can be a component of a
water treatment kit.
[0218] In certain embodiments, the membrane transporter is
expressed on the mitochondrial membrane. In certain embodiments,
the membrane transporter is SMF1. In certain embodiments, the
vacuole transporter is CCC1. In certain embodiments, both membrane
transporter, SMF1, and vacuole transporter CCC1, can be used in
combination to uptake heavy metals such as cadmium. Compared to
wild-type, cadmium uptake increased by 10-fold. In certain
embodiments, SMF1 can be engineered to become sensitive to other
metals such as strontium, lead, and mercury. In certain
embodiments, conserved transmembrane domains can be identified
through global multi-alignments and mutagenized these portions to
create SMF1 libraries. These libraries can be tested against
different metals and assayed based on density changes because of
the mass increase due to metal accumulation.
[0219] In addition, to counter the toxicity effects of metal
uptake, plant phytochelatin synthase, TaPCS1 can be expressed to
increase metal tolerance. The combined expression of CCC1 and
TaPCS1 allows yeast to survive at 100 .mu.M cadmium, whereas
wild-type yeast dies at concentrations beyond 5 .mu.M.
[0220] In certain embodiments, the metal can be a divalent metal.
In certain other embodiments, the metal can be a transition
metal.
Strategy 3--Metal Conversion--Using Yeast'a Sulfur By-Products to
Precipitate Heavy Metals
[0221] Strategy 3 uses sulfur released from engineered yeast to
react with metals in solution.
[0222] A composition and a method for remediating a metal to treat
water or for mining a metal can include a cell including a
knocked-out enzyme required in sulfate-assimilation pathway, where
the cell has specificity for a metal.
[0223] In certain embodiments, enzymes required in the
sulfate-assimilation pathway can be knocked out to retard the
conversion of sulfates to thiol metabolites allowing a build-up of
hydrogen sulfide precursors. In addition, nutrient sources such as
cysteine and methionine can be varied to affect the production rate
and quantity of produced hydrogen sulfide.
[0224] Preliminary studies show sulfur accumulating up to 1 mM in
culture which readily reacts with copper, cadmium, and lead.
Investigation under TEM shows that reacted metal sulfides form
consistently sized nanoparticles in the range of 20-50 nm on the
yeast cell wall. Particles are easily purified by enzymatically
digesting the cell wall, pelleting the cellular debri, and
collecting the supernatant. Specifically, purified CdS particles
exhibit a unique excitation and emission wavelength at 395 and 430
respectively, characteristic of quantum dots in that size-range.
The purity and quality of yeast generated quantum dots can be
confirmed using X-ray diffraction and TEM. In parallel, various
conditions such as pH, media composition, and strain-type can be
tested to understand the effect on metal sulfide formation with
respects to size, crystallinity, quantity, and monodispersity.
[0225] In certain embodiments, the metal can be a divalent metal.
In certain other embodiments, the metal can be a transition
metal.
1. Strategy 1--Cell Surface Capture
[0226] A paper by Ruta et. al. functionalized the yeast surface
with hexapeptides to capture a range of common divalent metals such
as nickel, copper, iron, etc. In addition, cells with displaying
metal binding proteins tend to be more metal tolerant, as metals
captured extracellularly are prevented from entering the cell body.
See Robert Wysocki and Markus J Tamas. How Saccharomyces cerevisiae
copes with toxic metals and metalloids. FEMS microbiology reviews,
34 (6):925-951, 2010, Lavinia Liliana Ruta, Ralph Kissen, Ioana
Nicolau, Aurora Daniela Neagoe, Andrei Josa Petrescu, Atle M Bones,
and Ileana Cornelia Farcasanu. Heavy metal accumulation by
Saccharomyces cerevisiae cells armed with metal binding
hexapeptides targeted to the inner face of the plasma membrane.
Applied Microbiology and Biotechnology, pages 1-15, 2017, and Oscar
N Ruiz, Derry Alvarez, Gloriene Gonzalez-Ruiz, and Cesar Torres.
Characterization of mercury bioremediation by transgenic bacteria
expressing metallothionein and polyphosphate kinase. BMC
biotechnology, 11 (1):82, 2011, each of which is incorporated by
reference in its entirety. However, current cell surface binding
capacities, which are in the .mu.M range, still cannot compete with
ion-exchange due to their extremely low capture capacity and poor
capture to cell weight ratio. See M A Barakat. New trends in
removing heavy metals from industrial wastewater. Arabian Journal
of Chemistry, 4 (4):361-377, 2011, and P Stathi, I T Papadas, A
Tselepidou, and Y Deligiannakis. Heavy-metal uptake by a high
cation-exchange-capacity montmorillonite: The role of permanent
charge sites. Global nest, 12 (3):248-255, 2010, each of which is
incorporated by reference in its entirety. Therefore, the number of
binding sites per cell is the greatest limiting factor that limits
the effectiveness of cell surface display.
1.1.Using Multiplier Proteins to Increase Metal Uptake Capacity
[0227] One method to address the limitation of cell surface display
is to display multiple repeats of the same binding protein on a
single displayed unit. Going one step further, one can instead
aggregate proteins onto the yeast surface to create a metal capture
network. This mechanism of aggregating proteins onto the yeast
surface can be achieved by a combination of displaying and
secreting so called "multiplier" proteins, proteins that
oligomerize to themselves. For example, an engineered yeast strain
can display a single multiplier protein, and the same, or another
yeast strain, can secrete the same protein into the media. In the
presence of metal the secreted proteins can oligomerize and
inevitably anchor to the protein displayed on the yeast surface,
thereby forming an aggregated network. This network anchored on the
yeast surface effectively multiplies the expression level of the
typically single displayed protein on the yeast surface (FIG. 2).
FIG. 2 shows one cell displaying a protein oligomer, and another
(or the same cell) can secrete protein monomers that aggregate onto
the displayed protein. Linkers fused with other proteins of
interest (POI) can be appended to the oligomers to tailor the
network's metal binding properties.
[0228] In certain embodiments, the multiplier protein used in this
strategy can be glutamine synthetase (GS for short; PDB: 2GLS), a
bacterial protein which has been studied for its role in glutamate
and glutamine synthesis. One unique property that has been somewhat
overlooked is its ability to aggregate in a structurally unique
pattern in the presence of divalent metals (FIGS. 3A-3C). In FIGS.
3A-3C, top images are rendered structures of GS from
crystollographic data. Bottom are TEM images of GS units as well as
aggregates. See Bennett M Shapiro and E R Stadtman. [130] glutamine
synthetase (Escherichia coli). Methods in enzymology, 17:910-922,
1970, which is incorporated by reference in its entirety. The rate
of aggregation and the degree of oligomerization is dependent on
exposure time and concentration of metal.
[0229] As a pilot-study, glutamine synthetase was overexpressed and
purified from BL21 bacteria cells and added at 100 .mu.M to the
medium containing yeast displaying the same protein. Aggregation
was visible by eye as well as examined using scanning electron
microscopy. The effect of aggregation on metal uptake was
quantitatively measured using inductively coupled plasma (ICP).
Species such as nickel, iron, and copper had uptakes of 5-10 mM
whereas cobalt, lead, and cadmium ranged from 1-5 mM given 1 OD600
of cells in synthetically defined media (CSM). The difference in
binding capacity between species like iron and copper to the
heavier and larger atoms such as lead and cadmium could be due to
different GS binding affinities. In FIG. 5, top image shows (tubes
labeled left to right) solution of yeast, and yeast with 10 mM Cu,
10 mM Co, and 10 mM Cd. The right image shows the same conditions
but with the addition of 100 .mu.M GS. Bottom figure shows metal
uptake percentage quantified using ICP.
[0230] Without cells, glutamine synthetase alone is an effective
metal binder. But with the addition of yeast, the aggregated
network aggregates and sinks, simply due to the yeast's higher
density allowing for easy separation from treated waters. Another
advantage is that the aggregate can now be packed onto filtration
or chromatography columns as membrane filters are typically
sub-micron to micron which can easily exclude yeasts. So rather
than handling liquids of culture and proteins, one can instead
package these aggregated complexes in filtration columns that are
easy to handle and use.
1.2. Tethering Metal Binding Proteins/Motifs for Increase Metal
Specificity
[0231] To further augment the binding capacity of this multiplier
system, additional proteins containing metal binding domains can be
fused onto GS to increase binding capacity and tailor for metal
specificity. In certain embodiments, proteins such as plant
metallothioneins can be used because of their low molecular weight
and high metal binding affinity as well as their multiple binding
domains (between 7-14). See Christopher Cobbett and Peter
Goldsbrough. Phytochelatins and metallothioneins: roles in heavy
metal detoxification and homeostasis. Annual review of plant
biology, 53 (1):159-182, 2002, which is incorporated by reference
in its entirety. Also, plant metallothioneins have a high affinity
for other metals such as mercury and strontium that do not bind to
GS. See Gerald Henkel and Bernt Krebs. Metallothioneins: Zinc,
cadmium, mercury, and copper thiolates and selenolates mimicking
protein active site features-structural aspects and biological
implications. Chemical reviews, 104 (2):801-824, 2004, and Ivo
Fabrik, Jiri Kukacka, Jiri Baloun, Ivo Sotornik, Vojtech Adam,
Richard Prusa, David Vajtr, Petr Babula, and Rene Kizek.
Electrochemical investigation of strontium-metallothionein
interactions-analysis of serum and urine of patients with
osteoporosis. Electroanalysis, 21 (3-5):650-656, 2009, each of
which is incorporated by reference in its entirety.
[0232] In certain embodiments, these plant metallothioneins (MTs)
can be appended to the N' terminus of GS, as GS's main aggregating
domain is located at the C' terminus (determined by analyzing the
crystal structure and using predicitve algorithms such as TANGO
(see Ana-Maria Fernandez-Escamilla, Frederic Rousseau, Joost
Schymkowitz, and Luis Serrano. Prediction of sequence-dependent and
mutational effects on the aggregation of peptides and proteins.
Nature biotechnology, 22 (10):1302, 2004, which is incorporated by
reference in its entirety). By tethering plant MTs on the GS
multiplier protein system, metal binding capacities can be at least
doubled, and more so binding affinities can now favor heavier
elements such as cadmium, lead, and mercury.
1.3. Additional Embodiments to Improve Secretion Yields
[0233] The biggest limitation of the current multiplier protein
system is the low expression levels of GS, both with respects to
display and secretion. Even with codon optimization, GS is
displayed on less than 20% of cells (FIG. 6), and secretion levels
are barely detectable via Western Blot (not shown). FIG. 6 shows 2D
histogram of FACS data observing fluorescently tagged N'-terminus
HA tag (FITC,.lamda.ex=488 nm) and C'-terminus Myc tag
(CY5,.lamda..sub.ex=647 nm).
[0234] Western blots of cell lysate show two bands with equal
intensity, one with the correct molecular weight of GS, and the
larger being GS+signal peptide. Therefore, there are two
inefficiencies for GS export, the first being proper cleavage of
the signal pep-tide, and the second is the transport out of the
cell after peptide cleavage.
[0235] In certain embodiments, S. cerevisiae's
.alpha.-mating-factor can be used as a signal peptide.
[0236] In certain embodiments, other mating factors such as AGA1/2
and EXG1 can be used to improve yield as they are processed through
different secretion pathways possibly allowing easier passage and
folding. See Lars Ellgaard, Maurizio Molinari, and Ari Helenius.
Setting the standards: quality control in the secretory pathway.
Science, 286 (5446):1882-1888, 1999, and Gunnar von Heijne. The
signal peptide. Journal of Membrane Biology, 115 (3):195-201, 1990,
each of which is incorporated by reference in its entirety.
[0237] In certain embodiments, the expression process can be
conducted at 20.degree. C. (or lower) to improve proper GS folding
in order to avoid the cell's unfolded protein response which
destroys poorly folded proteins in the endoplasmic reticulum, and
is often a major problem for secreting heterologous proteins in
yeast. See David Ron and Peter Walter. Signal integration in the
endoplasmic reticulum unfolded protein response. Nature reviews.
Molecular cell biology, 8 (7):519, 2007, and Dagang Huang, Patrick
R Gore, and Eric V Shusta. Increasing yeast secretion of
heterologous proteins by regulating expression rates and
post-secretory loss. Biotechnology and bioengineering, 101
(6):1264-1275, 2008, each of which is incorporated by reference in
its entirety.
[0238] In other embodiments, GS can be expressed in Pichia
Pastoris, a commonly used yeast host strain for heterologous
expression of prokaryotic and eukaryotic proteins that has a well
defined secretory pathway.
[0239] In other embodiments, a different multiplier besides GS can
be used. The identity of the protein is not a major concern just as
long as it can be sufficiently displayed and secreted.
[0240] Another survey can be done through the literature and the
protein data bank (www.resb.org/pdb/home/home.do, which is
incorporated by reference in its entirety). To identify a suitable
candidate, a multiplier protein must have well characterized and
controllable aggregating properties. These aggregating properties
must remain when fused to another protein (e.g. plant MTs) either
at the N' or C' terminus and secrete more efficiently than GS.
2. Strategy 2--Metal Uptake
[0241] Plants have evolved a unique ability to tolerate heavily
contaminated soils, specifically heavy metals such as cadmium,
arsenic, chromium, etc. See Nicoletta Rascio and Flavia
Navari-Izzo. Heavy metal hyperaccumulating plants: how and why do
they do it? and what makes them so interesting? Plant science, 180
(2):169-181, 2011, and Majeti Narasimha Vara Prasad and Helena
Maria de Oliveira Freitas. Metal hyperaccumulation in plants:
biodiversity prospecting for phytoremediation technology.
Electronic journal of biotechnology, 6 (3):285-321, 2003, each of
which is incorporated by reference in its entirety. Researchers
have attributed this unique ability to a combination of
hyper-active metal transporters and a variety of metal-binding
proteins that uptake and guard against metal poisoning. See Stephan
Clemens, Michael G Palmgren, and Ute Kramer. A long way ahead:
understanding and engineering plant metal accumulation. Trends in
plant science, 7 (7):309-315, 2002, which is incorporated by
reference in its entirety. Strategy 2 utilizes a parallel mechanism
to that of plants by endowing yeast strains with hyperactive
membrane and vacuole transporters, as well as promiscuous metal
binding proteins to create yeast hyperaccumulators that internalize
large amounts of metals.
2.1. Expressing Relevant Transporters and Proteins to Achieve
Hyperaccumulating Activity
[0242] Much like plants, the requirements for metal
hyperaccumulation in yeast are: 1) membrane metal transporters; 2)
vacuole transporters; and 3) metal sequestration proteins to
increase metal tolerance.
[0243] Out of 16 transporters screened, yeast SMF1 was chosen
because of its well-studied mechanism of action in addition to its
ability to transport a variety of divalent metals. See P Courville,
R Chaloupka, and M F M Cellier. Recent progress in
structure-function analyses of nramp proton-dependent metal-ion
transporters this paper is one of a selection of papers published
in this special issue, entitled csbmcbmembrane proteins in health
and disease. Biochemistry and Cell Biology, 84 (6):960-978, 2006,
which is incorporated by reference in its entirety. most metal
transporters are heavily regulated by proteases and ubiquitinases
to balance the concentration of intracellular metals. See Elina
Nikko, James A Sullivan, and Hugh R B Pelham. Arrestin-like
proteins mediate ubiquitination and endocytosis of the yeast metal
transporter smf1. EMBO reports, 9 (12):1216-1221, 2008, and Steven
Lam-Yuk-Tseung, Gregory Govoni, John Forbes, and Philippe Gros.
Iron transport by nramp2/dmt1: ph regulation of transport by 2
histidines in transmembrane domain 6. Blood, 101 (9):3699-3707,
2003, each of which is incorporated by reference in its entirety.
The major SMF1 ubiquitination ligase, BSD2, was deleted (see Xiu
Fen Liu and Valeria Cizewski Culotta. Post-translation control of
nramp metal transport in yeast role of metal ions and the bsd2
gene. Journal of Biological Chemistry, 274 (8):4863-4868, 1999,
which is incorporated by reference in its entirety) with no signs
of compromising yeast health. In addition, lysine residues K33,34
in SMF1 were mutated to arginines to destroy the primary
ubiquitination sites of SMF1 that are recognized by other
ubiquitination ligases. See Elina Nikko, James A Sullivan, and Hugh
R B Pelham. Arrestin-like proteins mediate ubiquitination and
endocytosis of the yeast metal transporter smf1. EMBO reports, 9
(12):1216-1221, 2008, which is incorporated by reference in its
entirety. The engineered SMF1 is referred to as SMF1* (or
SMF1-star).
[0244] Finally, a vacuole transporter was chosen by co-expressing
selected ones with SMF1* and choosing the vacuole transporter that
enhanced the uptake of cadmium. In FIG. 7, blue is DAPI nuclear
stain. SMF1 appended with a V5 tag was stained with AlexaFluor 488
(green), and CCC1 appended with a ag tag was stained with AlexFluor
647 (red). The best performing candidate was CCC1, normally a
Fe.sup.2+ and Mn.sup.2+ transporter, which showed more than 3-fold
increase in cadmium uptake (FIG. 8).
2.2. Mutating and Screening of SMF1 for Changes in Metal
Specificity and Selectivity
[0245] One of the biggest limitations to rationally engineering
metal transporters is the lack of structural information due to
crystallization difficulty and the inability to reconstitute
membrane-like environments ex-vivo. See Elisabeth P Carpenter,
Konstantinos Beis, Alexander D Cameron, and So Iwata. Overcoming
the challenges of membrane protein crystallography. Current opinion
in structural biology, 18 (5):581-586, 2008, which is incorporated
by reference in its entirety. Therefore, most structure-to-function
information is obtained via meticulous and often times tedious
point mutations in hypothesized residues. Many of these studies
have identified significant transmembrane domains, yet most of the
results lead to a loss of function. See P Courville, R Chaloupka,
and M F M Cellier. Recent progress in structure-function analyses
of nramp proton-dependent metal-ion transporters this paper is one
of a selection of papers published in this special issue, entitled
csbmcbmembrane proteins in health and disease. Biochemistry and
Cell Biology, 84 (6):960-978, 2006, Steven Lam-Yuk-Tseung, Gregory
Govoni, John Forbes, and Philippe Gros. Iron transport by
nramp2/dmt1: ph regulation of transport by 2 histidines in
transmembrane domain 6. Blood, 101 (9):3699-3707, 2003, and J M
Arguello. Identification of ion-selectivity determinants in
heavy-metal transport p1b-type atpases. The Journal of membrane
biology, 195 (2):93-108, 2003, each of which is incorporated by
reference in its entirety.
[0246] Fortunately, SMF1 has significant homology and phylogenetic
relationships with a large class of divalent transporters, namely
Nramps (natural resistance-associated macrophage proteins) and DMTs
(divalent metal transporters). See Ute Kramer, Ina N Talke, and
Marc
[0247] Hanikenne. Transition metal transport. FEBS letters, 581
(12):2263-2272, 2007, which is incorporated by reference in its
entirety. Because of this, researchers have discovered conserved
regions that are necessary for SMF1 function. Through sequence
analysis researchers have hypothesized that transmembrane domains
1-4, 5-6, and 9 are responsible for discriminating between and
facilitating metal transport.
[0248] In order to further narrow the number of significant
domains, a global multi-alignment analysis with 81 of the closest
ranked members similar to SMF1 was performed. The 81 members were
filtered from a potential list of >14,000 sequences queried
based on name searches from Uniprot (http://www.uniprot.org/) by
using Clustal Omega (http://www.ebi.ac.uk/Tools/ msa/clustalo/).
The 81 members were globally aligned and spans of homology were
quantified using Shannon entropy (Equation 1) which is a simple and
direct metric for identifying conserved protein regions. See
William S J Valdar. Scoring residue conservation. Proteins:
structure, function, and bioinformatics, 48 (2):227-241, 2002,
which is incorporated by reference in its entirety.
H ( X ) j = i = 0 n P ( x i ) log 2 P ( x i ) ( 1 )
##EQU00002##
[0249] Rows of aligned sequences (i) are scored per residue with
respects to the queried sequence (j) by calculating the probability
(P) of that residue's appearance in the global alignment according
to the Shannon entropy function. The lowest entropic score (most
conserved) regions were transmembrane domain 1 and 6. Both domains
were amplified using error-prone PCR and homologously recombined
into SMF1 to generate a library of mutants.
Developing High Throughput Screening of Mutated SMF1 Library
[0250] Mutants were screened by subjecting libraries to 100 .mu.M
metal ions (Me.sup.2+) in culture and fractionated based on density
changes. Changes in density were used to assess differences in
metal uptake, as the uptake of metals imparts additional mass to
the cell. See William H Grover, Andrea K Bryan, Monica Diez-Silva,
Subra Suresh, John M Higgins, and Scott R Manalis. Measuring
single-cell density. Proceedings of the National Academy of
Sciences, 108 (27):10992-10996, 2011, which is incorporated by
reference in its entirety. Cells were separated using density
gradient centrifugation; the furthest migrated layers in the
density gradient were manually selected, re-plated, sequenced, and
quantitatively tested for metal uptake using ICP. These rounds were
repeated 3 times for cadmium, and are ongoing for elements such as
strontium and lead.
[0251] In FIG. 9, SMF1 was mutagenized using error-prone PCR and
assayed for metal uptake using density gradient centrifugation.
Cells that uptake the most metals migrate furthest to the bottom.
Cells were isolated with a syringe, then plated, picked, sequenced,
and finally confirmed for metal uptake using ICP.
2.3. Increasing Metal Tolerance & Survival
[0252] Along with hyperactive metal transporters, plants can
tolerate unusually high metal concentrations by arming themselves
with a variety of metal binding proteins, namely glutathiones and
metallothioneins. See Christopher Cobbett and Peter Goldsbrough.
Phytochelatins and metallothioneins: roles in heavy metal
detoxification and homeostasis. Annual review of plant biology, 53
(1):159-182, 2002, which is incorporated by reference in its
entirety. Plants augment this layer of defense by oligomerizing
glutathiones into phytochelatins which act as a network to
internalize metals into compartmentalized areas. Id. Clemens et.
al. found that a common wheat phytochelatin synthase, TaPCS1,
dramatically enhanced metal tolerance when expressed in yeast. See
Stephan Clemens, Eugene J Kim, Dieter Neumann, and Julian I
Schroeder. Tolerance to toxic metals by a gene family of
phytochelatin synthases from plants and yeast. The EMBO journal, 18
(12):3325-3333, 1999, which is incorporated by reference in its
entirety. Performing a similar experiment, a constitutive
overexpression of TaPCS1 directly cloned from wheat to wild-type
yeast improved cadmium tolerance by almost 20-fold (FIGS. 10A-10B).
FIG. 10A shows a diagram illustrating plant phytochelatin TaPCS1 to
enhance metal tolerance I yeast. See Clemens, S., Kim, E. J.,
Neumann, D. & Schroeder, J. I. Tolerance to toxic metals by a
gene family of phytochelatin synthases from plants and yeast. The
EMBO Journal 18, 3325-3333 (1999), which is incorporated by
reference in its entirety. In FIG. 10B, cultures were grown in
increasing amounts of cadmium levels (0-100 .mu.M) and measured
periodically at OD600. Growth rate k were extracted from curves
using the logistic growth function. The left figure are wild-type
yeast, the right figure are wild-type yeast constitutively
expressing wheat phytochelatin synthase TaPCS1.
2.4. Additional Embodiments
[0253] In certain embodiments, SMF1 transporters can be created to
be selective not only to cadmium, but also for additional metals
such as strontium, lead, mercury, etc. by focusing on filtering out
libraries of SMF1 mutants based on increase metal uptake, metal
selectivity (KD) and specificity between metals. For example,
interference experiments can determine whether mutants can uptake
cadmium in the presence of excess manganese (the preferred metal).
Likewise, an experiment for metal selectivity can be executed to
characterize titration curves in order to determine the KD response
of a given metal using colorimetric assays or ICP.
Uptake of Negatively Charged Metal Compounds
[0254] Not all heavy metals are positively charged, there exist
polyatomic metal compounds in the negative state such as chromate
(CrO.sub.4.sup.2-) and arsenate (AsO.sub.4.sup.2-) which are
acutely toxic, however unrecognized by SMF1. See A D Dayan and A J
Paine. Mechanisms of chromium toxicity, carcinogenicity and
allergenicity: review of the literature from 1985 to 2000. Human
& experimental toxicology, 20 (9):439-451, 2001, and Michael F
Hughes. Arsenic toxicity and potential mechanisms of action.
Toxicology letters, 133 (1):1-16, 2002, each of which is
incorporated by reference in its entirety. Yet, there exist
permeases, much like metal transporters, that facilitate the flux
of basic nutrients such as sulfates (SO.sub.4.sup.2-) and
phosphates (PO.sub.4.sup.2-) into the cell. See Bruno Andre. An
overview of membrane transport proteins in Saccharomyces
cerevisiae. Yeast, 11 (16):1575-1611, 1995, which is incorporated
by reference in its entirety. Given the structural similarity
between chromates and sulfates, and between arsenates and
phosphates (FIGS. 12A-12B), it may be possible to "hijack" these
permeases for chromate and arsenate uptake. As a preliminary
experiment, both sulfate permeases Sul1, and Sul2 were tested for
chromate uptake. As hypothesized, chromate uptake was elevated in
yeast expressing Sul1, Sul2, but not to the degree at which
overexpressing wild-type SMF1 uptakes divalent metals. One
explanation is that chromate is much more acutely toxic than
cadmium; at concentrations above 20 .mu.M yeast die and no longer
transport metals. Another explanation is that permeases are
selective enough to discern between sulfates and chromates. Or
perhaps the sulfate concentration in yeast media overwhelms the
transport of chromate.
Applications in Mining
[0255] Admittedly, cellular uptake of heavy metals has the least
per cell uptake capacity ratio than Strategy 1. However, a main
advantage of metal transporters is that they are more sensitive to
low amounts of metals, and are more metal specific. Besides SMF1,
there exist other selective metal transporters such as CTR1
(copper), ZRT1 (zinc), and FRE1 (iron). These transporters are able
to recognize and uptake .mu.M to nM amounts of metals despite the
presence of other more concentrated ions such as sodium and calcium
naturally found in growth media. See Stephan Clemens, Michael G
Palmgren, and Ute Kramer. A long way ahead: understanding and
engineering plant metal accumulation. Trends in plant science, 7
(7):309-315, 2002, which is incorporated by reference in its
entirety. This metal specific uptake can be capitalized to mine
useful metals from waste water in addition to providing a mechanism
for metal removal. With the growing demand of electronically
relevant metals such as lithium, noble metals (gold, silver,
platinum), and rare-earth metals, mining operations are becoming
exceedingly more dangerous and harmful to the environment. See
Gavin Hilson. Pollution prevention and cleaner production in the
mining industry: an analysis of current issues. Journal of Cleaner
Production, 8 (2):119-126, 2000, which is incorporated by reference
in its entirety. Likewise, metal extraction typically takes several
rounds of heating and smelting to purify a single element, which is
labor intensive and costly. See Jirang Cui and Lifeng Zhang.
Metallurgical recovery of metals from electronic waste: A review.
Journal of hazardous materials, 158 (2):228-256, 2008, and AM
Alfantazi and R R Moskalyk. Processing of indium: a review.
Minerals Engineering, 16 (8):687-694, 2003, each of which is
incorporated by reference in its entirety. Given these limitations
it may be possible to engineer yeast with specialized metal
transporters to act as mining agents that harvest and concentrate
scarce amounts of metals from environmental sources under ambient
conditions.
[0256] One example would be mining for lithium, noble metals (such
as gold, silver, platinum) and rare-earth metals (such as cerium
(Ce), dysprosium (Dy), erbium (Er), europium (Eu), gadolinium (Gd),
holmium (Ho), lanthanum (La), lutetium (Lu), neodymium (Nd),
praseodymium (Pr), promethium (Pm), samarium (Sm), scandium (Sc),
terbium (Tb), thulium (Tm), ytterbium (Yb) or yttrium (Y)). A
report by MIT projects that lithium shortages will occur by 2050,
yet some researchers acknowledge that this can be avoided if the
ocean were mined instead. See Richard Martin, As Demand for Lithium
Grows, the Race to Extract It Intensifies, 2015, which is
incorporated by reference in its entirety. Despite an overall
massive quantity, approximately 1.5 trillion tons, lithium
concentrations are low, ranging from 0.1-1 ppm (sub micromolar),
and differentiating between the more abundant salt content makes
lithium mining almost impossible. See Ernest E Angino and Gale K
Billings. Lithium content of sea water by atomic absorption
spectrometry. Geochimica et Cosmochimica Acta, 30 (2):153-158,
1966, which is incorporated by reference in its entirety. A similar
situation poses a challenge for rare-earth mining. Ironically, some
rare-earths are more abundant than more known elements such as
cobalt and manganese, yet they are typically difficult to extract
because of the overwhelming presence of iron, copper, and nickel
compounds in mining ores. See Xiaoyue Du and Thomas E Graedel.
Global in-use stocks of the rare earth elements: a first estimate.
Environmental science & technology, 45 (9):4096-4101, 2011,
which is incorporated by reference in its entirety. Again, highly
specific yeast transporters could help differentiate rare-earths
from other elements and aid in the mining process. Yeast can
effectively act as concentrators, specializing and storing desired
metals of interest. Yeast can then be harvested and lysed to obtain
the stored metal. Afterwards, simpler and more straightforward
physicochemical techniques can be used to isolate and purify these
metals rather than having to smelt and reheat ores common of
traditional methods.
3. Strategy 3--Metal Conversion
[0257] The type of metal as well as its electronic state are
equally important in determining the metals' toxicity. For example,
Cr(VI) is highly mutagenic and acutely toxic, whereas Cr(III)
readily forms stable oxides and precipitates out of solution. See
Olga Muter, Aloizij s Patmalnieks, and Alexander Rapoport.
Interrelations of the yeast Candida utilis and cr (vi): metal
reduction and its distribution in the cell and medium. Process
Biochemistry, 36 (10):963-970, 2001, which is incorporated by
reference in its entirety. Therefore, it is just as important to
convert metals to more benign electronic states as it is to capture
and remove them from waste waters. However, what has limited
bio-facilitated conversion and reactions of heavy metals is the
burden of supplying electron-rich molecules (e.g. pyruvates and
NADP(H)s), which are themselves rarely free in the cell other than
for highly regulated biological processes. Even if these
biomolecules are present, the electrons must overcome a large
activation barrier for converting normally stable metal ions to a
more benign state.
3.1. Encouraging Sulfur Production in Yeast
[0258] Normally, cells are unable to process large amounts of
metals from the environment; however, there exist a unique class of
archaebacteria that can convert a select set of metals and organic
compounds to other electronic states (e.g. Fe.sup.2+Fe.sup.3+) for
metabolic purposes. See Derek R Lovley. Dissimilatory metal
reduction. Annual Reviews in Microbiology, 47 (1):263-290, 1993,
and Karrie A Weber, Laurie A Achenbach, and John D Coates.
Microorganisms pumping iron: anaerobic microbial iron oxidation and
reduction. Nature Reviews Microbiology, 4 (10):752-764, 2006, each
of which is incorporated by reference in its entirety. For example,
a class of bacteria, known as sulfate-reducins, obtains energy by
reducing sulfate (SO.sub.4.sup.2-) to hydrogen sulfide (H.sub.2S),
and in the process gain energetic electrons for other cellular
functions. Because of the production of hydrogen sulfide, these
sulfate-reducins are able to erode iron and copper comprised rocks,
and have unfortunately become a hazard to old concrete and metal
infrastructures due to accelerated sulfur corrosion. See Washington
A Hamilton. Sulphate-reducing bacteria and anaerobic corrosion.
Annual Reviews in Microbiology, 39 (1):195-217, 1985, which is
incorporated by reference in its entirety. However researchers have
deliberately used this phenomenon to remove rusted metals from old
mines and drains for cleaning purposes. See C Garcia, D A Moreno, A
Ballester, M L Blazquez, and F Gonzalez. Bioremediation of an
industrial acid mine water by metal-tolerant sulphate-reducing
bacteria. Minerals Engineering, 14 (9):997-1008, 2001, which is
incorporated by reference in its entirety. Given what is known
about the scarcity of electron rich species in the cell, sulfur
lends itself extremely well as a metal reactant that is
biologically generated. If production can be controlled, sulfur
could be utilized as a reliable source for heavy metal
remediation.
Effects of knockouts
[0259] Rather than using a difficult-to-culture sulfur-reducin,
Strategy 3 is focused on creating sulfur-producing yeast.
Surprisingly, the wine industry has studied the effects of yeast
related-sulfur production with respect to wine quality. For the
past century, winemakers have realized that over-fermentation, or
failing to supply sufficient nutrient sources, causes yeast to
produce a pungent smell during wine-making. See Carla S Thomas,
Roger B Boulton, Michael W Silacci, and W Douglas Gubler. The
effect of elemental sulfur, yeast strain, and fermentation medium
on hydrogen sulfide production during fermentation. American
journal of enology and viticulture, 44 (2):211-216, 1993, which is
incorporated by reference in its entirety. With the aid of recent
molecular biology techniques, researchers have discovered that due
to extreme culture conditions essential proteins in the sulfate
reducing pathway are either inhibited or denatured causing a
buildup of sulfide (H.sup.2-) precursors. See J H Swiegers and I S
Pretorius. Modulation of volatile sulfur compounds by wine yeast.
Applied Microbiology and Biotechnology, 74 (5):954-960, 2007, which
is incorporated by reference in its entirety.
[0260] Enzymes in the sulfate assimilation pathway were knocked out
to force a buildup of H.sub.2S (FIGS. 13A-13G). In FIGS. 13A-13G,
italicized enzymes were knocked out and screened for sulfur
production. Italicized and bolded enzymes are knockouts that
produced a detectable amount of sulfur. All others are necessary
enzymes that are required for sulfur metabolism. See Angela L
Linderholm, Carrie L Findleton, Gagandeep Kumar, Yeun Hong, and
Linda F Bisson. Identification of genes affecting hydrogen sulfide
formation in Saccharomyces cerevisiae. Applied and environmental
microbiology, 74 (5):1418-1427, 2008, Chien Huang, Miguel
Roncoroni, and Richard C Gardner. Met2 affects production of
hydrogen sulfide during wine fermentation. Applied microbiology and
biotechnology, 98 (16):7125-7135, 2014, and Chien-Wei Huang,
Michelle E Walker, Bruno Fedrizzi, Miguel Roncoroni, Richard C
Gardner, and Vladimir Jiranek. The yeast tum1 affects production of
hydrogen sulfide from cysteine treatment during fermentation. FEMS
yeast research, 16 (8), 2016, each of which is incorporated by
reference in its entirety. Knockouts such as .DELTA.MET2,17 and
.DELTA.HOM2 produced an observable amount of sulfur determined by
lead acetate strip and sulfur-displacement columns (FIG. 15A),
whereas some deletions such as .DELTA.CYS4 and .DELTA.SER1,2
produced cysteine and methionine auxotrophy. Multi-deletions (2
& 3 knockouts) produced sick strains that barely formed
colonies even on YPD (yeast peptone dextrose media), and therefore
were not tested.
Effects of Media Composition and Nutrients
[0261] The size and crystal properties of these nanoparticles can
be tuned by changing the nutritional content of the yeast culture,
primarily cysteine and methionine precursors, thereby changing the
production rate and timing of sulfur, which ultimately effects the
kinetics of particle growth. More so, if these particles are
properly made, they also have fluorescent properties (FIG. 32B),
which makes production of these complex particles autonomous,
tunable, and cheap.
[0262] FIG. 14 shows a more quantitative detection method is to use
columns that change color at a given height as a function of sulfur
production. Rich culture sources such as YPD allowed all knockouts
to grow, yet some produced little-to-no sulfide. Compared to CSM
media (complete supplement mixture, which lacks cysteine and
contains minimal methionine), .DELTA.MET2,17 and .DELTA.HOM2
produced sulfide at levels up to 100 ppm (FIG. 15A); however
mutants such as .DELTA.CYS4 and .DELTA.SER1,2 failed to grow due to
cysteine or methionine auxotrophy.
[0263] Supplementing cysteine to CSM rescues .DELTA.CYS4 allowing a
production of 102 ppm whereas .DELTA.MET2,17 and .DELTA.HOM2
produce almost half. Likewise, adding more methionine to CSM
reduces .DELTA.MET2,17 and .DELTA.HOM2 production by half. An
explanation for the nutrient effects on sulfide production is that
the addition of cysteine or methionine eliminates the stress for
thiol biosynthesis which reduces sulfide production (FIG. 15C).
Conversely, the addition of cysteine allows .DELTA.CYS4 to grow,
and since .DELTA.CYS4 is the biggest roadblock for cysteine
synthesis (hence the auxotrophy), the sulfate pathway terminates at
hydrogen sulfide gas which is thereby released.
3.2. Yeast Induced Metal Sulfide Precipitation
[0264] Metal sulfides have an extremely low solubility constant in
solutions pH adjusted between 4-10. See DigitalAnalysisCo. Heavy
Metal Reduction from Industrial Wastewater Streams, 2016, which is
incorporated by reference in its entirety. Because of this,
industrial chemical precipitation sometimes uses sulfur to treat
highly contaminated waste water. However, the volatility and
storage of sulfur becomes a hazard, so many industrial sites
instead opt for sodium hydroxide. See Eddy Metcalf. Wastewater
engineering: Treatment, disposal, reuse, metcalf & eddy. Inc.,
McGraw-Hill, New York, 2003, which is incorporated by reference in
its entirety. Sulfide-producing yeast, however, is not burdened by
practical issues of sulfur management because the yeast itself can
be easily packaged and stored. Storage of sulfur simply requires
the storage of yeast that produces it. Furthermore, sulfide
production can be easily regulated depending on the demand by
controlling nutrient conditions and gene expressions in the sulfate
assimilation pathway.
Producing Cd, Cu Particles
[0265] .DELTA.CYS4, .DELTA.HOM2, and .DELTA.MET2,17 mutants are
able to precipitate approximately 1 mM Cu.sup.2+ and 100 .mu.M
Cd.sup.2+ in CSM (.DELTA.CYS4 cultures supplemented with
cysteine).
[0266] Cross-sectional examination of metal precipitated cells
using cryo-sectioning and TEM show that CuS and CdS particles
precipitate on the cell wall. Simple lysis of the cell wall
releases these particles which consistently range between 20-50 nm
in diameter.
3.3. Using Yeast Display to Control Metal Sulfide Formation
[0267] Crystalline and structured metal sulfides are valuable for
their applications in electronics, material fabrication, and
optics. See Jagadese J Vittal and Meng Tack Ng. Chemistry of metal
thio- and seleno-carboxylates: precursors for metal
sulfide/selenide materials, thin films, and nanocrystals. Accounts
of chemical research, 39 (11):869-877, 2006, which is incorporated
by reference in its entirety. Compounds such as CdS, PbS, ZnS are
routinely used for optics and quantum dot synthesis. See A Mews, A
Eychmuller, M Giersig, D Schooss, and H Weller. Preparation,
characterization, and photophysics of the quantum dot quantum well
system cadmium sulfide/mercury sulfide/cadmium sulfide. The Journal
of Physical Chemistry, 98 (3):934-941, 1994, which is incorporated
by reference in its entirety.
[0268] Yeast are able to uniformly precipitate CuS, CdS, ZnS, PbS,
and HgS; however, the mechanism is currently unknown, yet may be
due to interactions with the cell wall. In certain embodiments,
particle formation can controlled via changing the surface
chemistry of the cell wall by displaying peptides using yeast
display. There are already known peptides that can mineralize the
formation of CdS and ZnS, which has been done on the M13
bacteriophage coat protein by the Belcher Lab. See Chuanbin Mao,
Christine E Flynn, Andrew Hayhurst, Rozamond Sweeney, Jifa Qi,
George Georgiou, Brent Iverson, and Angela M Belcher. Viral
assembly of oriented quantum dot nanowires. Proceedings of the
National Academy of Sciences, 100 (12):6946-6951, 2003, which is
incorporated by reference in its entirety.
Designing Metal Sulfide Biomineralization Libraries
[0269] A straightforward approach is to take known metal sulfide
mineralization peptides and test for mineralization. However, the
current literature is limited to only a few peptides that
facilitate mineralization in extreme buffer conditions not amenable
to yeast cultures (e.g. high pH, low salt content, and typically
hazardous reducing agents). Moreover, these peptides solely focus
on CdS, PbS, or CuS which can already be formed with
sulfur-producing yeast.
[0270] Therefore, new metal sulfide biomineralization peptides have
been self-generated by creating a yeast display library with
degenerate sequence (NDN-NNK).sub.(8,12,16) (SEQ ID NOS 1-3)
(subscripts denoting repeats) that are biased towards cysteine,
histidines, glutamic and aspartic acid residues. These libraries
are inserted into a yeast display expression vector with the
canonical yeast display AGA1 and AGA2 cassette (plasmid named
pYAGA). Such libraries can be generated and mutants can be visually
screened based on metal sulfide color changes either in cultures or
on plates supplemented with the metal of interest (FIG. 18). More
quantitative screening can be investigated using ICP and TEM.
3.4. Additional Embodiments
[0271] In certain embodiments, sulfide biomineralization yeast can
be engineered and culture conditions can be optimized to control
for metal sulfide particle formation.
[0272] In certain embodiments, yeast-synthesized metal sulfides can
be used for either materials or electronics.
Investigating pH Effects on Fe, Pb, Zn, Etc. Particle Formation
[0273] Adding 100 .mu.M sodium sulfide (Na.sub.2S) to yeast media
readily precipitates copper, cadmium, as well as iron, lead,
mercury and zinc. However, metals other than copper or cadmium are
very difficult to precipitate with sulfur-producing yeast despite a
similar buildup of produced sulfide. One hypothesis is that the
rate of sulfur production is too slow. Meaning, the rate of sulfide
escaping from the media as a gas is faster than the rate of
reaction between metal and sulfide for the more soluble metals such
as iron and zinc. And as a function of time, after 12-16 hours of
yeast growth the culture pH can drop to 2, at which the pH is
outside the permissible solubility range of most metal sulfide
formation (FIG. 19). In FIG. 19, solubility/dissociation products
and calculations were taken from online resources. See Renata
Benova, Danica Melicher ikova, and Peter Tom ik. Calculation of
conditional equilibrium in serial multiple precipitation of metal
sulfides with hydrogen sulfide stream generated from sodium
sulfide: A didactic tool for chemistry teaching. Quimica Nova, 39
(6):765-769, 2016, which is incorporated by reference in its
entirety. Therefore, a slow sulfide production rate can accumulate
slower than the drop in pH and prevent metal sulfide formation. A
possible solution is to simply buffering yeast media to >pH 7
prior to inoculation to maintain the pH of the media above 4.
[0274] In summary, three unique strategies can utilize yeast for
heavy metal remediation. The first employs cell surface display in
combination with protein cell secretion to aggregate multiple
repeats of the same protein on the yeast surface. This strategy
improves upon conventional yeast display capture by multiplying the
number of protein binding domains thereby increasing metal capture
capacity up to 1-10 mM. The second strategy focuses on metal
internalization by engineering metal transport systems at the cell
membrane and vacuole. Using engineered SMF1 membrane transporter
and CCC1 vacuole transporter, uptake of cadmium increases 10-fold
when compared to WT. In certain embodiments, SMF1 can be being
further modified to become sensitive to other relevant metals such
as strontium, lead, and mercury. Finally, the third strategy uses
hydrogen sulfide by-products from the yeast sulfate-assimilation
pathway to react and precipitate heavy metals from solution. In
certain embodiments, the formation of metal sulfide particles can
be controlled with yeast displayed biomineralization peptides, or
modulating hydrogen sulfide production rate, in order to obtain
useful metal sulfide nanoparticles such as quantum dots. Strategies
1-3 can be combined to provide a powerful method for heavy metal
remediation. Current ideas to synergistically combine the various
strategies of metal binding, transport, and mineralization are
shown in FIGS. 20A-20C.
[0275] These strategies can be used to build a platform in which
industries and the public can accessibly and cheaply purify water.
What makes yeast such an attractive platform is the ease to
engineer and rapidly test better performing strains, and this can
only get simpler with more advanced genetic engineering tools. In
addition yeast circumvents many limitations hampering current
physicochemical processes, such as renewability, cost, and
production of secondary waste. Already the industry to cheaply
scale the production of yeast exists because of the beer and
pharmaceutical industry. See Argyro Bekatorou, Costas Psarianos,
and Athanasios A Koutinas. Production of food grade yeasts. Food
Technology and Biotechnology, 44 (3):407-415, 2006, which is
incorporated by reference in its entirety. Likewise, the food
industry has establish protocols to handle, transport, and store
yeast for consumer use, so there exist a feasible entry way for
this technology to enter the public market. The methods disclosed
here can be combined with already established yeast production
infrastructures in order to provide yeast at low costs. Current
infrastructures from the beer, pharma, and food industries can be
utilized to create another avenue in which yeast can remediate
toxic materials from industrial processes such as mining, chemical
spills, and manufacturing runoff.
EXAMPLES
Yeast as a Sequestration Agent
[0276] A yeast strain that endogenously displays the AGA1 &
AGA2 surface proteins was designed by constructing a DNA construct
containing the AGA1 and AGA2 sequence with a strong constitutive
promoter (GPO) and a canonical transcription terminator (CYC1). In
addition, the AGA2 is followed by a protein of interest (POI)
flanked by restriction sites NheI and XhoI (for cloning purposes)
and a N'-HA and C'-Flag tag (for expression studies). Finally, the
construct is appended to a TRP1 autotrophic marker for positive
selection of transformed yeast. The POI are a family of plant
metallothioneins (MT1A-4A) and the yeast endogenous metallothionein
(CUP1). Metallothioneins are known to have strong affinities for
copper, zinc, mercury, and lead. See Robinson, Nigel J., et al.
"Plant metallothioneins," Biochemical Journal 295.Pt 1 (1993): 1.
Yeast display of the AGAI & AGA2 constructs is used to express
multiple copies of these metallothioneins (50,000-100,000 copies;
see Boder. Eric T., and K. Dane Wittrup, "Yeast surface display for
screening combinatorial polypeptide libraries," Nature
biotechnology 15.6 (1997): 553-557, which is incorporated by
reference in its entirety) on a single yeast surface to act as a
metal binding domains.
TABLE-US-00001 TABLE 1 Table listing the 4 families of
metallothioneins from Arabidopsis thaliana and the yeast
metallothionein (CUP1) used for yeast display SEQ ID MT gene
Protein Sequence NO MT1A MADSNCGCGSSCKCGD 4 SCSCEKNYNKECDNCS
CGSNCSCGSNCNC MT2A MSCCGGNCGCGSGCKC 5 GNGCGGCKMYPDLGFS
GETTTTETFVLGVAPA MKNQYEASGESNNAEN DACKCGSDCKCDPCTC K MT3
MSSNCGSCDCADKTQC 6 VKKGTSYTFDIVETQE SYKEAMIMDVGAEENN
ANCKCKCGSSCSCVNC TCCPN MT4A MADTGKGSSVAGCNDS 7 CGCPSPCPGGNSCRCR
MREASAGDQGHMVCPC GEHCGCNPCNCPKTQT QTSAKGCTCGEGCTCA SCAT CUP1
NIFSELINFQNEGHEC 8 QCQCGSCKNNEQCQKS CSCPTGCNSDDKCPCG
NKSEETKKSCCSGK
[0277] Data presented in this report are with respects to plant
Arabidopsis thaliana metallothionein MT2A for conciseness and
because all MTs tested show extremely similar results. Yeast
display expression of MT2A increases Cu(II) uptake by 4-5 fold
compared to WT which is >100 times higher than the US
Environmental Protection Agency (EPA) actionable level of 1.3 ppm
for allowable copper concentrations in drinking water. See
file:///C:/Users/GeorgeSun/AppData/Roaroing/Zotero/Zotero/Profiles/wc-
3qz9ge.default/zotero/storage/S.
regulated-drinking-water-contaminants.html, which is incorporated
by reference in its entirety. In addition, MT2A yeast display
strains are able to tolerate and thrive in high copper
concentrations of about 16-20 mM, whereas WT die below 5 mM.
[0278] The plant metallothionein protein can be further engineered
or evolved and screened for greater metal binding efficiency,
capacity, and/or selectivity via high-throughput genetic
engineering and screening methods such as flow cytometry.
Yeast as a Metal Absorber
[0279] At a different perspective, the yeast's entire intracellular
volume can be viewed as a sequestration locale. Several yeast
strains that over-express a unique metal transporter (Table 2) were
created by following similar DNA design strategies as described
above (FIG. 21). See, Hall, J. L., and Lorraine E. Williams,
"Transition metal transporters in plants," Journal of experimental
botany 54.393 (2003): 2601-2613, which is incorporated by reference
in its entirety. Each metal transporter gene is controlled by a
GAL1 inducible promoter and followed by a V5 epitope tag (for
staining purposes) and a CYC1 transcription terminator.
[0280] Of the expressed strains, metal uptake for Cu(II), Zn(II),
and Mn(II) were tested.
TABLE-US-00002 TABLE 2 A list of most prominent metal transporters
involved in yeast. Name Description ZRT1 High-affinity zinc
transport protein ZRT2 Low-affinity zinc transport protein ZRT3
Transports zinc from storage in the vacuole to the cytoplasm ZAP1
Involved in zinc ion homeostasis by zinc responsive transcriptional
regulation CTR1 Required for high affinity copper (probably reduced
CuI) transport into the cell CTR2 Provides bioavailable copper via
mobilization of vacuolar copper stores and export to the cytoplasm
CTR3 Required fur high affinity copper (probably reduced CuI)
transport into the cell FRE1 Metalloreductase responsible for Fe3+
and Cu2+ salts FRE2 Metalloreductase responsible for Fe3+ and Cu2+
salts FTR1 Permease for high affinity iron uptake FET3 Iron
transport multicopper ferroxidase required for Fe2+ ion high
affinity uptake. Required to oxidize Fe2+ and release it from the
transporter. Essential component of copper-dependent iron transport
FET4 Required for Fe2+ ion low affinity uptake BSD2 Required for
homeostasis of heavy metal ions such as cadmium, cobalt and copper.
Under manganese-replete conditions facilitates trafficking of SMF1
and SMF2 metal transporters to the vacuole where they are degraded
SMF1 High-affinity manganese transporter involved in manganese
uptake from the extracellular environment. Contributes also to
cellular accumulation of other divalent metal ions such as cadmium,
cobalt, copper, iron and nickel SMF2 High-affinity manganese
transporter involved in mobilizing manganese from vesicular stores
in conditions of low manganese ion concentrations
Recycling & Conversion of Toxic Metals Using Yeast Display
[0281] A metal's toxicity is based on its oxidation state and the
molecular compound in which it is in. For example, Cr(VI),
specifically chromates (CrO.sub.4.sup.2-, Cr.sub.2O.sub.7.sup.2-)
is considered highly mutagenic and acutely toxic, however Cr(III)
is insoluble in water and is overall less reactive.
[0282] Therefore, it is equally important to capture heavy metals
as it is to convert and possibly recycle these metals to a more
benign and usable form.
[0283] Organisms have already discovered methods to convert metals
from an unfavorable state to a more favorable one. Specifically,
cytochromes found in dissimilatory metal reducing bacteria are
known to transfer electrons to heavy metals as terminal electron
accepters in order to generate an electrochemical gradient for the
production of chemical energy. See Lovley, Derek R. "Dissimilatory
metal reduction." Annual Reviews in Microbiology 47.1 (1993):
263-290, which is incorporated by reference in its entirety. These
proteins all contain a porphyrin cofactor in which a chelated metal
facilitates the transfer of electrons from a metabolic substrate
(i.e. conversion of NAD(P)H, formate, lactate, or pyruvate, etc.)
to heavy metals from the environment. A similar mechanism is being
pursued in yeast in which yeast cytochromes, CYC1 & 7, are
displayed on the surface in hopes of facilitating electron transfer
between environmental heavy metal and the cytochrome's heme group.
An increase in redox potential of yeast displayed CYC1/7 was
observed as evidence for the possibility of facilitated heavy metal
reduction.
[0284] Biologically catalyzed metal reduction can be further
pursued to create microbial fuel cells in which an organism,
typically bacterial catalyzes the oxidation or reduction of an
organic or inorganic matter to generate current. The same principal
to reduce metals using yeast displayed cytochromes can
simultaneously be harnessed to generate and store current ill a
fuel cell.
Metal-Contaminated Yeast After Water Treatment
[0285] Metal-contaminated yeast filter packets can be handled many
different ways after water treatment. The first is to reuse or
recycle the filter packet by removing the captured heavy metal. The
most direct method to release captured metals is to gently wash the
yeast filter packet for metal removal at reduced pH (pH 3-4). The
second method is to enzymatically treat the yeast with proteases to
cleave the protein-metal complexes off the cells. After separation,
yeast can be air-dried, stored, and later reconstituted when
another batch needs to be cultured (FIG. 25). In FIG. 25, the first
stage (green layer) provides an easy method to culture and
propagate stocks of yeast. Yeast can be grown indefinitely given
enough space and nutrients, or stored and consolidated in packets
for later use. When yeast are needed (orange layer), cultures can
be aliquoted from the main stock and added into the remediation
filter packet. Contaminated waters can be manually pumped or washed
over the filter packet to capture any metal contaminants. From
here, yeast can be separated from the captured metal by washing at
reduced pH, enzymatic, or salt treatment (blue layer). Yeast can be
reused, composted, or autolyzed as a nutrient source for subsequent
culture growth. Captured metals should be thrown out to dedicated
waste sites or delivered to industries or government agencies that
can recycle the metals.
[0286] A drop in strain integrity was observed after remediation,
such as slower growth rates, incomplete separation of contaminants,
or strain mutations, yeast can be lysed and then biodegraded. Lysis
simply requires salted water, approximately a few hundred
millimolar (6-30 grams per liter of water) (see Huh, G.-H. et al.
Salt causes ion disequilibrium-induced programmed cell death in
yeast and plants. Plant J. 29, 649-659 (2002), which is
incorporated by reference in its entirety), to create a hypertonic
environment in which yeast undergo autolysis. Hypertonic solutions
swell and eventual burst the yeast cell releasing enzymes and
proteases causing cellular degradation. Autolysis effectively
reduces yeast biomass into yeast extract, a rich blend of basic
nutrients such as amino acids and nitrogen sources that can feed
future cultures of yeast. See Chae, H. J., Joo, H. & In, M.-J.
Utilization of brewer's yeast cells for the production of
food-grade yeast extract. Part 1: effects of different enzymatic
treatments on solid and protein recovery and flavor
characteristics. Bioresour. Technol. 76, 253-258 (2001), and
Tanguler, H. & Erten, H. Utilisation of spent brewer's yeast
for yeast extract production by autolysis: The effect of
temperature. Food Bioprod. Process. 86, 317-321 (2008), each of
which is incorporated by reference in its entirety. Yeast extract
is not toxic (as yeast extract is commonly used as a food additive
for flavoring). There also exist commercial food brands based on
yeast extract such as Vegemite and Marmite.
Target Heavy Metals
[0287] Two categories of heavy metals can be considered. The first
category is divalent elemental metals such as lead (Pd.sup.2+),
mercury (Hg.sup.2+), cadmium (Cd.sup.2+), etc. The second category
is polyatomic and organic metals such as chromium, which is
typically found as chromate (CrO.sub.4.sup.2-); arsenic, which is
typically found as arsenate (AsO.sub.4.sup.3-); and organomercury,
which is found in numerous states with methyl functional groups
(e.g., CH.sub.3-Hg.sup.+).
[0288] The first category was successfully demonstrated in terms of
enhancing uptake capacity, specificity, and ability to sequester
and compartmentalize elemental metal contaminants. However, for
polyatomic and organic metal compounds, due to their different
chemistries, differing yeast metabolic pathways, and obviously the
difference in charge and valency, the strategies to address
elemental metal capture need to be adjusted. Yeast permeases, in
particular sulfate (SO.sub.4.sup.2-) and phosphate
(PO.sub.4.sup.3-) permeases, can be used to uptake chromate
(CrO.sub.4.sup.2-) and arsenate (AsO.sub.4.sup.3-), respectively,
since the structural similarity between the two species would allow
for direct pumping in of the metal oxide counterpart.
Hyperaccumulation of chromate and arsenate using two sulfate
permease genes (Sul1 and Sul2) and are in a position to further
engineer and optimize the system. The other two strategies, yeast
surface capture and mineralization are still being engineered to
accommodate this second category of targets. This technology can be
used for water treatment in areas such as Flint as well as other
neglected areas poisoned by industrial runoff and mining
effluent.
Safety for Drinking Water Applications
[0289] An easy to follow and robust method for water treatment that
is adaptable to geographical location, types of water sources, user
level of expertise, in addition to withstanding common user modes
of failure can be provided. The mode of operation is to filter
water through a yeast packet and funnel the flow through into a
water container. Alternatively, the packet can be submerged in the
treated water (much like a sponge) where the water can then be
collected.
[0290] To ensure robustness of yeast filter packet, treated waters
can be strained in a size-exclusion filter to remove particulate
and yeast. Commonly used and available size-exclusion filters with
0.2-0.5 .mu.m (see Corning sterile filtration guide, available at:
www.corning.com/media/worldwide/cls/documents/CLS-FIL-004%20REV4%20DL.pdf
(accessed: 30 Mar. 2017), which is incorporated by reference in its
entirety) pore sizes are small enough to segregate yeast from
entering the water.
[0291] Also, the strains are engineered to be autotrophic, that is
they lack the capacity to produce essential nutrients for growth,
typically amino acids such as tryptophan, histidine, leucine etc.
Unless supplied by the user (during culture), yeast cannot survive
and will eventually die if outside of filter.
[0292] The engineered strains are able to flocculate given an
external stimulus. The packet can contain factors that suppress
flocculation. In the event that the yeast reside outside the
filter, then without access to the flocculation suppressing
factors, flocculation would occur, which induces yeast
precipitation and automatic removal from the water.
[0293] A variety of pH- and metal-tolerant biodegradable hydrogels
can encapsulate and secure yeast in a contained filter packet.
These packets can then be surrounded in a semi-porous membrane
(e.g. similar to dialysis tubing) to allow diffusion of treated
water into the packet, yet disallow movement of larger molecules
beyond a given molecular size cutoff. See Ma, Y. et al. Effects of
nanoplastics and microplastics on toxicity, bioaccumulation, and
environmental fate of phenanthrene in fresh water. Environ. Pollut.
219, 166-173 (2016), Gimpel, J., Zhang, H., Davison, W. &
Edwards, A. C. In Situ Trace Metal Speciation in Lake Surface
Waters Using DGT, Dialysis, and Filtration. Environ. Sci. Technol.
37, 138-146 (2003), and Nolan, A. L., Mclaughlin, M. J. &
Mason, S. D. Chemical Speciation of Zn, Cd, Cu, and Pb in Pore
Waters of Agricultural and Contaminated Soils Using Donnan
Dialysis. Environ. Sci. Technol. 37, 90-98 (2003), each of which is
incorporated by reference in its entirety. The hydrogels in the
packet are meant to be degraded so that metals can be stripped and
segregated from the yeast after use. Yeast can then be harvested
for re-inoculation or degradation as described in FIG. 25.
[0294] If the packet degrades after successive water treatments,
the semi-porous membrane adds another barrier of selection against
particles dissolving into the water. The dialysis tubing, most
likely made of nitrocellulose or a cellulose monomer, is typically
resistant to many chemicals and pH ranges and has long shelf and
usage lifetimes. See SnakeSkin Dialysis Tubing, available at:
tools.thermofisher.com/content/sfs/manuals/MAN0011339_SnakeSkin_Dialy_Tub-
ing_UG.pdf (accessed: 30 Mar. 2017), and Spectra Cellulose Dialysis
Membrane, available at:
http://www.spectrumlabs.com/lit/420x10116x000.pdf (accessed: 30
Mar. 2017), each of which is incorporated by reference in its
entirety.
[0295] Although yeast has been engineered to secrete a variety of
proteins for commercial and pharmaceutical purposes, native yeast
has a limited secretome. See Choi, J. et al. Fungal Secretome
Database: Integrated platform for annotation of fungal secretomes.
BMC Genomics 11, 105 (2010), which is incorporated by reference in
its entirety. Most secreted proteins are typically mating factors
or pheromones needed to communicate with other cells during haploid
mating events. See Loumaye, E., Thorner, J. & Catt, K. J. Yeast
mating pheromone activates mammalian gonadotrophs: evolutionary
conservation of a reproductive hormone? Science 218, 1323-1325
(1982), which is incorporated by reference in its entirety. If this
is any cause for concern, these pathways can be easily knocked out
to limit the amount of factors secreted. Secretion may not be a
concern given that beer is essentially the collection and reduction
of yeast supernatant (these strains will not be fermented, so no
production of sugars or alcohols should exist in the filtered
water).
[0296] To prevent incomplete metal capture, preliminary on-site
measurements can be performed to see how this yeast filter packet
capture capacity scales with the amount of metal contaminants in
the local waters to be treated. Given an approximate estimate of
local contaminant concentrations, different sizes or densities of
yeast packets can be used to efficiently remove all contaminants in
one treatment cycle. Alternatively, multiple yeast packets can be
used in-tandem per treatment for increased capture capacity.
[0297] In the treatment pipeline, a metal sensing packet can be
introduced. A packet containing yeast can colorimetrically or
fluorescently respond to metal contaminants. For example, the
packet can contain yeast that has a green fluorescent protein (GFP)
downstream of a metal-inducible promoter (FIG. 26). These packets
can be separated from the remediation packet, but can still follow
the culturing and usage pipeline outlined in FIG. 25. Promoters
include those that control metallothionein and glutathione
transcription (CUP1, MTF-1, etc.), which can be used to control GFP
expression. See Saydam, N., Adams, T. K., Steiner, F., Schaffner,
W. & Freedman, J. H. Regulation of Metallothionein
Transcription by the Metal-responsive Transcription Factor MTF-1
IDENTIFICATION OF SIGNAL TRANSDUCTION CASCADES THAT CONTROL
METAL-INDUCIBLE TRANSCRIPTION. J. Biol. Chem. 277, 20438-20445
(2002), and Ecker, D. J. et al. Yeast metallothionein function in
metal ion detoxification. J. Biol. Chem. 261, 16895-16900 (1986),
each of which is incorporated by reference in its entirety.
Wastewater Remediation Versus Drinking Water
[0298] Wastewater remediation is of great interest, particularly
mining and agricultural runoff. However, the most pressing issue in
developing areas and economically disadvantaged communities is the
inaccessibility of safe drinking water. The disclosed method can
provide a renewable platform to continuously grow and use water
remediation agents (yeast) that can empower communities to clean
their own waters. Accessible and usable in DIY packets with yeast
can be grown and maintained in a user-friendly manner in many
geographical regions.
[0299] Yeast versus sulfate-generating bacteria (to precipitate
metalloid sulfides) Yeasts are easier to grow, have shorter
doubling times, and have an extensive toolkit for molecular biology
engineering. In addition, yeast strains that are able to produce
sulfur require the same nutrients as wild-type strains, as the
deletion of the sulfate-assimilation pathway does not perturb any
other metabolic pathway.
[0300] In comparison to sulfur-generating bacteria, commonly found
in the families of Desulfobacterales, Desulfovibrionales and
Syntrophobacterales (see Muyzer, G. & Stams, A. J. M. The
ecology and biotechnology of sulphate-reducing bacteria. Nat. Rev.
Microbiol. 6, 441-454 (2008), which is incorporated by reference in
its entirety), these strains are very difficult to grow and
engineer compared to yeast. These cultures require anaerobic
conditions, meaning oxygen is lethal to their growth and require
controlled anaerobic chambers devoid of oxygen with precise control
of humidity and temperature. In addition, production of sulfur
requires additional nutrients that are otherwise not needed for
commonly used bacteria and yeast strains, such as propionate, high
sulfate concentrations, or fermentable lactate and ethanol. See
Muyzer, G. & Stams, A. J. M. The ecology and biotechnology of
sulphate-reducing bacteria. Nat. Rev. Microbiol. 6, 441-454 (2008),
and H Kadota & Ishida, and Y. Production of Volatile Sulfur
Compounds by Microorganisms. Annu. Rev. Microbiol. 26, 127-138
(1972), each of which is incorporated by reference in its entirety.
Even under the best growth conditions, the pathway and control of
sulfur production in sulfur-generating bacteria are relatively
unclear and still require further investigation. See Schippers, A.
& Sand, W. Bacterial Leaching of Metal Sulfides Proceeds by Two
Indirect Mechanisms via Thiosulfate or via Polysulfides and Sulfur.
Appl. Environ. Microbiol. 65, 319-321 (1999), and Friedrich, C. G.,
Rother, D., Bardischewsky, F., Quentmeier, A. & Fischer, J.
Oxidation of Reduced Inorganic Sulfur Compounds by Bacteria:
[0301] Emergence of a Common Mechanism? Appl. Environ. Microbiol.
67, 2873-2882 (2001), each of which is incorporated by reference in
its entirety. Understanding this pathway is further hindered by the
difficulty of genetically engineering sulfur-generating strains, as
they offer few avenues for genetic manipulation compared to more
evolved bacteria and yeast strains. Strain engineering in bacteria
usually requires horizontal gene transfer of a shuttle vector from
a well-defined and engineerable host such as bacteria; this added
layer of complexity overall slows the engineering pipeline for
improved strain performance. See Dodsworth, J. A. et al.
Interdomain Conjugal Transfer of DNA from Bacteria to Archaea.
Appl. Environ. Microbiol. 76, 5644-5647 (2010), which is
incorporated by reference in its entirety.
[0302] To summarize, the engineered yeast strains require minimal
culture effort, grow at ambient temperature with limited impact on
ambient air conditions, and produce appreciable quantities of
sulfur. In a head-to-head comparison with respect to sulfur
production, the engineered strains can produce 55.+-.8 ppm of
sulfur per culture (12 hours of growth in an Erlenmeyer flask),
whereas depending on the bacteria strain, nutrient source, and
culturing method (flask, fermentation chamber, bioreactor, etc.)
production of sulfur can range from 7.5 to 67 ppm (see Jong, T.
& Parry, D. L. Removal of sulfate and heavy metals by sulfate
reducing bacteria in short-term bench scale upflow anaerobic packed
bed reactor runs. Water Res. 37, 3379-3389 (2003), which is
incorporated by reference in its entirety) in 12 hours. Yeast is
favored because of its ease of use, known sulfur pathway, and
controllability of sulfur production.
Scalability and Preliminary Cost Analysis of DIY Yeast Packets
[0303] Beer industry can be used as a reference on the economics of
yeast production and cite academic literature on bioseparation
processes and distribution for evaluating the scalability and cost
of the DIY yeast packets. The United States alone produced 55
billion gallons of beer in 2012. See The Economics of Craft
Beer|SmartAsset.com. SmartAsset (2017), available at:
smartasset.com/credit-cards/the-economics-of-craft-beer (accessed:
2 Apr. 2017), which is incorporated by reference in its entirety.
Typically, 2-10 billion yeast cells are needed to ferment a single
gallon of beer (see Baker, D. A. & Kirsop, B. H. Rapid Beer
Production and Conditioning Using a Plug Fermentor. J. Inst. Brew.
79, 487-494 (1973), which is incorporated by reference in its
entirety), making yeast production estimated at approximately 24
thousand tons of biomass per year (see Bryan, A. K., Goranov, A.,
Amon, A. & Manalis, S. R. Measurement of mass, density, and
volume during the cell cycle of yeast, Proc. Natl. Acad. Sci. 107,
999-1004 (2010), which is incorporated by reference in its
entirety). These yeasts are either recycled for use in another
production batch or discarded. These numbers do not consider the
production of yeast for consumer and pharmaceutical goods like
bread, dried-yeast packets, and therapeutic compounds which is on
the same order of magnitude as the beer industry. What this means
is that yeast is already a cheap, scalable, and consumer friendly
type of microorganism that can be similarly scaled and distributed
for water remediation purposes.
[0304] In typical bioprocessing settings (averaged for
pharmaceutical applications) the cost of raw yeast is approximately
$4 dollars per kilogram. See Harrison, R. G., Todd, P., Todd, P.
W., Rudge, S. R. & Petrides, D. P. Bioseparations Science and
Engineering. (Oxford University Press, 2015), which is incorporated
by reference in its entirety. Ingredients to maintain cultures such
as glucose, yeast extract, amino acids, and trace elements go for
$3 dollars per kilogram of total material. Id. In the lab,
typically 1 million cells are seeded per mL of yeast in 1 liter
cultures. Therefore, the cost to start a culture is 16 cents per L
culture. In the laboratory setting, and especially in batch and
fermentation processes, yeast can undergo several doublings per
inoculum. A final 1-liter culture may have as much as
1.sup.10-1.sup.11 cells in 16 hours (experimentally determined).
Therefore, the final cost of yeast per cost of raw material is most
likely an order of magnitude lower than what is calculated. With
respects to the cost of yeast needed per packet, assuming
contamination levels of up to 100 .mu.M or more (equivalent to ten
to a thousand times higher than EPA standards for certain metals;
see EPA. Wastewater Technology Fact Sheet Chemical Precipitation
(2000), available at:
//nepis.epa.gov/Exe/ZyPDF.cgi/P1001QTR.PDF?Dockey=P1001QTR.PDF
(accessed: 5 Jan. 2017), which is incorporated by reference in its
entirety), 1.sup.10 yeast cells or less would be required to
completely purify a 1 liter solution (based on experimental
results). Therefore, a packet containing 1.sup.10 cells would
require a fraction of a liter, meaning the cost to purify water can
start as low as a few pennies per liter of drinking water.
[0305] Therefore, the most expensive aspect of setting up a culture
is the cost of equipment, transportation, and packaging. Breaking
down the cost further, a pound (454 g) of freeze- or active-dried
yeast for DIY brewing or bread making costs 5-30 dollars depending
on the quality of yeast (i.e. number of surviving cells, QC
testing, brand identification, etc.). Home brewing kits (typically
sized as 5 gallons) can go for $30 to $300 dollars, again depending
on the quality of the brand. See Association, H. A. Is Homebrewing
Cheaper than Store-Bought Beer? American Homebrewers Association
(2015), available at:
www.homebrewersassociation.org/news/is-homebrewing-cheaper-than-store-bou-
ght-beer/(accessed: 2 Apr. 2017), which is incorporated by
reference in its entirety. Therefore, to culture 1 L of yeast costs
roughly $5-10 dollars on the low end. However, the startup cost can
be lowered by using standard culturing flasks, simple ingredients,
and the ability to freeze-dry one's own cultures to self-propagate
cultures for future use.
Large-Scale Comparison of DIY Yeast Packets Versus Physicochemical
Methods
[0306] Whether synthesizing resins, adsorption filters, or
electrochemical substrates, the basic processing pipeline is as
follows (see Harrison, R. G., Todd, P., Todd, P. W., Rudge, S. R.
& Petrides, D. P. Bioseparations Science and Engineering.
(Oxford University Press, 2015), and Jr, W. D. C. & Rethwisch,
D. G. Fundamentals of Materials Science and Engineering: An
Integrated Approach. (John Wiley & Sons, 2012), each of which
is incorporated by reference in its entirety): (1) reactors and
synthesis, Primary recovery--solid and phase separation, (2)
intermediate recovery--ultrafiltration, evaporation, reverse
osmosis, etc., (3) final purification--crystallization, solvent/pH
exchange, chromatography, etc., (4) quality control (QC)--size,
structure, chemical composition/characterization, etc., and (5)
packaging and storage--typically requires technical handling and
storage. Whereas bioprocessing of yeast requires: (1) continuous
bioreactor, (2) cell separation--filtration or centrifugation of
cellular debris, (3) QC--routine genotyping, and (4) packaging and
storage--freeze-dried, air-dried, etc.
[0307] Yeast has advantages as it avoids complex synthesis steps,
requires a simple isolation process, and QC reduces to simple
strain genotyping to guarantee selection of engineered yeast.
Moreover, iteration becomes more feasible as molecular biology
engineering requires simple cloning technologies, such as DNA oligo
design, polymerase chain reaction (PCR), and transformations, which
can cost as little as a few dollars per experiment. See Guthrie, C.
& Fink, G. R. Guide to yeast genetics and molecular and cell
Biology: Part C. 351, (Gulf Professional Publishing, 2002), which
is incorporated by reference in its entirety. For physicochemical
processes, however, an entire synthesis pipeline may need to be
adjusted to accommodate changing reaction conditions, and possibly
entire facilities may need to be retro-fitted in case of
incompatible reaction steps.
[0308] When compared with the current resin technology
(chromatography), resins require chemical functionalization of
polystyrene beads whose chemistries can be quite complex, making
optimization of highly specified and selective resin groups
difficult and slow. See
[0309] Kentaro Tashiro, Modular synthesis of metal-organic complex
arrays containing precisely designed metal sequences, available at:
globalscience.berkeley.edu/sites/default/files/15-modularmoca.pdf
(accessed: 2 Apr. 2017), which is incorporated by reference in its
entirety. Likewise, resin-based technology may not be amenable for
public use as storage conditions may differ per resin type and
functional group, and resins are not easily recycled if users do
not have technical knowledge of the precise chemistries and best
practices for handling. Therefore, resins may actually contribute
to secondary waste if not properly managed.
[0310] On the other hand, yeast provides a better alternative for
resin water treatment, especially in the form of yeast display.
Surface protein expression is easily and greatly tunable on the
genetic level while maintaining identical culturing conditions for
un-engineered, or other engineered yeast strains. Likewise, yeast
can be re-used or stored for later use as described in the sections
above. But if yeast were to be discarded, they are biodegradable
and would not contribute to secondary waste.
[0311] Compared to the current adsorption filters, similar
arguments with resin technology, the manufacturing and chemistries
of adsorption filters and membranes inhibit this technology from
being widely distributed for public use. The first is the decline,
or scarcity of resource materials such as nanostructured materials
or carbon nanotubes. See Jong, T. & Parry, D. L. Removal of
sulfate and heavy metals by sulfate reducing bacteria in short-term
bench scale upflow anaerobic packed bed reactor runs. Water Res.
37, 3379-3389 (2003), and Stafiej, A. & Pyrzynska, K.
Adsorption of heavy metal ions with carbon nanotubes. Sep. Purif.
Technol. 58, 49-52 (2007), each of which is incorporated by
reference in its entirety. Likewise handling and recycling of
adsorption filters and membranes may be difficult to manage for
untrained users.
[0312] One of the most highly used physicochemical methods for
industrial waste treatment, chemical precipitation uses sacrificial
iron compounds or reactive hydroxyl or sulfur groups to precipitate
and remove metal complexes. See Charerntanyarak, L. Heavy metals
removal by chemical coagulation and precipitation. Water Sci.
Technol. 39, 135-138 (1999), which is incorporated by reference in
its entirety. For scaled industrial use chemical precipitation
costs are relatively cheap: approximately $0.05-$0.2 per liter of
water in the US. See Ozturk, I., Altinbas, M., Koyuncu, I., Arikan,
0. & Gomec-Yangin, C. Advanced physico-chemical treatment
experiences on young municipal landfill leachates. Waste Manag. 23,
441-446 (2003), which is incorporated by reference in its entirety.
However, this comes with hazardous pH ranges of 10-12 and handling
of several grams of chemicals per liter. Additionally, sulfur is
becoming a more prominent player in chemical precipitation for its
speed and reactivity; however, chemical storage of sulfide in the
form of sodium sulfide, iron sulfide, or sulfuric acid precursor is
incredibly dangerous to handle and should be avoided in public
hands. See Charerntanyarak, L. Heavy metals removal by chemical
coagulation and precipitation. Water Sci. Technol. 39, 135-138
(1999), which is incorporated by reference in its entirety.
[0313] The production of hydrogen sulfide from a biological source
is more benign than direct chemical precipitation. There is no need
for storage of precursor chemicals for sulfide production, as the
culture already contains the nutrients needed for yeast to
metabolize. So if sulfur production were to be controlled, yeast
can be easily moved from the media and idled in another buffer
source or stored for future use. Finally, the sulfur content
remains in solution and those that do become volatile and evaporate
into the atmosphere where the concentration dramatically reduces to
safe levels below EPA standards and is not a prominent safety
concern. See EPA. Public Health Statement--Hydrogen Sulfide,
available at: www.atsdr.cdc.gov/toxprofiles/tp114-cl-b.pdf
(accessed: 2 Apr. 2017), and By Scott
[0314] Simonton, P. D. & Oct. 3, 2007. Human Health Effects
from Exposure to Low-Level Concentrations of Hydrogen
Sulfide--Occupational Health & Safety, available at:
ohsonline.com/articles/2007/10/human-health-effects-from-exposure-to-lowl-
evel-concentrations-of-hydrogen-sulfide.aspx (accessed: 2 Apr.
2017), each of which is incorporated by reference in its
entirety.
Regionally Specific, Personally Customized Deployable DIY Yeast
Packets for Heavy Metal Remediation from Contaminated Water
[0315] The engineered yeast can be packaged into user-friendly and
economical units that can be deployed in areas in need of heavy
metal remediation to provide strains of yeast that are regionally
tailored so that users can simply grow and maintain their own
batches of yeast for personal water remediation efforts.
Instructions along with an ease to use culture containers can
provide users a means to regularly grow their own yeast stocks for
routine and self-sufficient water purification.
[0316] Yeast cell surface display of plant-based metal-binding
proteins, known as metallothioneins (MTs), is analogous to the
physicochemical ion-exchange technique. Four families of plant MTs
(MT1A-MT4A in FIG. 27B) as well as the yeast endogenous
metallothionein (CUP1) were expressed on the yeast surface and
assayed for Cu.sup.2+ uptake (the native ligand preferred by the
metallothionein families) (FIG. 27A). The AGA1 and AGA2 (purple)
domains were used to express peptides or proteins with metal
binding domains to remove contaminants from waters (orange
circles). Expression of MTs increased uptake capacity of Cu.sup.2+
by 3-4 fold compared to wild-type yeast (FIG. 27B). In addition,
expression of MTs increased tolerance of copper concentration in
solution by 3-4 fold (FIG. 27C).
[0317] Given modest estimates of yeast density and expression
levels, the upper limit of yeast display capture is in the
submillimole range per gram of dry weight. On the other hand,
synthetic ion-exchange resin capacities are in the 10
.sub..mu.mol-10 mmol of metal per gram of material (see
[0318] Barakat, M. A., New Trends in Removing Heavy Metals from
Industrial Wastewater. Arabian Journal of Chemistry 2011, 4 (4),
361-377, and Stathi, P.; Papadas, I. T.; Tselepidou, A.;
Deligiannakis, Y., Heavy-Metal Uptake by a High
Cation-Exchange-Capacity Montmorillonite: The Role of Permanent
Charge Sites. Global NEST Journal 2010, 12 (3), 248-255, each of
which is incorporated by reference in its entirety), up to 1-3
orders of magnitude greater than that demonstrated with the
metallothionein yeast display experiments (FIG. 27B). Despite an
overall lower binding capacity, an advantage of using yeast display
as a method for metal capture is the ability to engineer highly
specific metal-binding domains for extremely toxic metals, such as
mercury, while avoiding nonspecific saturation from common ions
such as Na, Ca, Mg, etc.
[0319] The shortcomings of traditional yeast cell surface display
can be overcome, while retaining the ability to engineer heavy
metal specificity, by the use of externally secreted multiplier
proteins, which greatly increase the number of metal-binding
domains available per gram of dry weight. MTs were then fused to a
multiplier protein, glutamine synthetase (GS). GS is a dodecahedron
protein with 12 subunits. Each subunit can be fused with a MT
appendage on the N'-terminus. Glutamine synthetase was engineered
to aggregate in response to a range of metals from zinc to cadmium
(FIG. 2). This system uses one strain to express a single copy of
the GS subunit on the yeast surface as the anchorage protein, with
another strain secreting the GS-MT complex. In the presence of
metals secreted GS-MT complexes being to aggregate and form on the
yeast surface producing a mesh network (FIG. 4). Binding capacities
are estimated to be enhanced by 2-3 orders of magnitude given the
increased amount of metal uptake than compared to singly displayed
MTs.
[0320] The plausibility of using yeast as a platform for metal
remediation becomes even more readily apparent when incorporating
multiplier proteins and proteins engineered for metal-specific
binding into the remediation strategy. Doing so increases capture
capacities to 1-10 mM, on par with synthetic ion-exchange resin
capacities, and allows for specific metal uptake (FIG. 5, lower).
In addition, due to their greater density, yeast bound to metals
can settle out of solution allowing for easy physical separation
from remediated waters (FIG. 5, upper).
[0321] In addition to possessing metal-binding proteins (MTs),
certain plants can uptake large quantities of metals, known as
hyperaccumulation. See Clemens, S.; Palmgren, M. G.; Kramer, U., A
Long Way Ahead: Understanding and Engineering Plant Metal
Accumulation. Trends in Plant Science 2002, 7 (7), 309-315, which
is incorporated by reference in its entirety. Such
hyperaccumulators often use metal transporter proteins for
subsequent compartmentalization into organelles called vacuoles or
bind metals using sequestration agents, primarily in the form of
phytochelatins or metallothionein proteins. See Song, W. Y.; Park,
J.; Mendoza-Cozatl, D. G.; Suter-Grotemeyer, M.; Shim, D.;
Hortensteiner, S.; Geisler, M.; Weder, B.; Rea, P. A.; Rentsch, D.;
Schroeder, J. I.; Lee, Y.; Martinoia, E., Arsenic Tolerance in
Arabidopsis Is Mediated by Two Abcc-Type Phytochelatin
Transporters. Proceedings of the National Academy of Sciences of
the United States of America 2010, 107 (49), 21187-21192, and
Cobbett, C.; Goldsbrough, P., Phytochelatins and Metallothioneins:
Roles in Heavy Metal Detoxification and Homeostasis. Annual Review
of Plant Biology 2002, 53, 159-182, each of which is incorporated
by reference in its entirety. However, because plants are
stationary, difficult to biologically engineer, and have a long
growth cycle, plants are not the best candidates to develop a
modular platform for a range of metal remediation tactics. Instead,
this strategy is to use plants as inspiration to engineer yeast for
heavy metal uptake and compartmentalization. There already exist
numerous yeast metal transporters similar to those of plants that
respond to various conditions such as pH, cofactors, and/or energy
resources. The same is also true for vacuole transporters that
secure toxins away from the yeast body.
[0322] Difficulty in predicting metal-binding regions and metal
specificity regions in these transporter proteins has previously
prevented rational design to attain better performance. See
Arguello, J. M., Identification of Ion-Selectivity Determinants in
Heavy-Metal Transport P-1b-Type ATPases. Journal of Membrane
Biology 2003, 195 (2), 93-108, which is incorporated by reference
in its entirety. Here, a screening method can use density changes
in the yeast cell as a direct measurement to qualitatively discern
metal uptake efficiency. Using established values for yeast cell
density and mass (see Bryan, A. K.; Goranov, A.; Amon, A.; Manalis,
S. R., Measurement of Mass, Density, and Volume During the Cell
Cycle of Yeast. Proceedings of the National Academy of Sciences of
the United States of America 2010, 107 (3), 999-1004, which is
incorporated by reference in its entirety), even metal uptake in
the hundreds of .mu.M can induce density changes up to 25%
depending on the molar mass of the metal. Density changes can be
discerned using density gradient separation techniques, such as
Percoll density centrifugation, which has a density resolution on
the order of 2-3%. See Ravnik, S. E.; Gage, S.; Pollack, S. B.,
Self-Generating Density Gradients of Percoll Provide a Simple and
Rapid Method That Consistently Enriches Natural-Killer Cells.
Journal of Immunological Methods 1988, 110 (2), 161-168, and
Childs, W. C.; Gibbons, R. J., Use of Percoll Density Gradients for
Studying the
[0323] Attachment of Bacteria to Oral Epithelial-Cells. Journal of
Dental Research 1988, 67 (5), 826-830, each of which is
incorporated by reference in its entirety. Using this density-based
method, libraries of transporters can be assayed and screened for
metal uptake efficiency given the direct physical change on yeast
density, and better performing strains can be selected visually and
filtered with increasingly more stringent density gradients (FIG.
28A).
[0324] Expression of the metal transporter protein of interest can
be increased, its degradation in regular cellular pathways can be
reduced, the transported metals can be shuttled into vacuoles for
containment, and finally yeast tolerance can be improved to
increased levels of metal accumulation (FIG. 29A). One such
transporter of interest is SMF1, a promiscuous divalent metal
transporter which has been observed to uptake a range of metals
such as manganese and cadmium. See Chen, X. Z.; Peng, J. B.; Cohen,
A.; Nelson, H.; Nelson, N.; Hediger, M. A., Yeast Smf1 Mediates
H+-Coupled Iron Uptake with Concomitant Uncoupled Cation Currents.
Journal of Biological Chemistry 1999, 274 (49), 35089-35094, which
is incorporated by reference in its entirety. SMF1, unlike most
other metal transporter proteins, benefits from 30-40 years of
sequence-function research. As such, SMF1 mutants were rationally
designed and compared for their metal uptake capacities. FIG. 28A
shows increased expression percentage as a function of SMF1
modifications, specifically conversion of lysine residues 33, 34 to
arginine (denoted with *) and a deletion of SMF1's degradation
protein BSD2 (denoted .DELTA.BSD2). With these rationally designed
modifications, cadmium accumulation was improved up to 4-fold
greater than that of wild-type yeast. Notably, the best performing
mutant so far is capable of exceeding 5 mg of cadmium per gram of
yeast dry weight, which is beyond the threshold for classifying
plants as cadmium hyperaccumulators (FIG. 29C). Modification of the
SMF1 metal transporter confers metal hyperaccumulator status. See
Rascio, N.; Navari-Izzo, F., Heavy Metal Hyperaccumulating Plants:
How and Why Do They Do It? And What Makes Them So Interesting?
Plant Science 2011, 180 (2), 169-181, which is incorporated by
reference in its entirety. Given the modularity of this approach,
the selection can be tailored for other metals of interest such as
radium and strontium, elements that are becoming increasingly
recognized as radioactive contaminants since the Fukushima incident
in 2014. See Iwahana, Y.; Ohbuchi, A.; Koike, Y.; Kitano, M.;
Nakamura, T., Radioactive Nuclides in the Incinerator Ashes of
Municipal Solid Wastes before and after the Accident at the
Fukushima Nuclear Power Plant. Analytical Sciences 2013, 29 (1),
61-66, which is incorporated by reference in its entirety. A suite
of yeast that can uptake a range of toxic elements with high
specificity can be developed in this way.
[0325] Another alternative strategy for metal remediation, in
addition to yeast cell surface display and yeast capture and
uptake, is to chemically reduce and precipitate metals from
wastewaters. Chemical precipitation is the most widely used method
for heavy metal remediation in industry, with hydrogen sulfide
being one of the most commonly used chemical precipitants. See Fu,
F. L.; Wang, Q., Removal of Heavy Metal Ions from Wastewaters: A
Review. Journal of Environmental Management 2011, 92 (3), 407-418,
and Metcalf, E.; Eddy, H. P.; Tchobanoglous, G., Wastewater
Engineering: Treatment, Disposal and Reuse. McGraw-Hill, New York
1991, each of which is incorporated by reference in its entirety.
On a tangential note, the wine industry discovered that yeasts are
able to produce an appreciable amount of hydrogen sulfide during
fermentation, causing wine to smell and taste pungent. See
Swiegers, J. H.; Pretorius, I. S., Modulation of Volatile Sulfur
Compounds by Wine Yeast. Applied Microbiology and Biotechnology
2007, 74 (5), 954-960, which is incorporated by reference in its
entirety. To inhibit sulfur production for better tasting wine,
Linderholm et al. and Huang et al. discovered that knockouts of
metabolic proteins, MET2, MET17 and CYS4, overproduce hydrogen
sulfide and are integral for complete sulfate metabolism for
production of amino acids such as cysteine. See Linderholm, A. L.;
Findleton, C. L.; Kumar, G.; Hong, Y.; Bisson, L. F.,
Identification of Genes Affecting Hydrogen Sulfide Formation in
Saccharomyces Cerevisiae. Applied and Environmental Microbiology
2008, 74 (5), 1418-1427, and Huang, C.; Roncoroni, M.; Gardner, R.
C., MET2 Affects Production of Hydrogen Sulfide During Wine
Fermentation. Applied Microbiology and Biotechnology 2014, 98 (16),
7125-7135, each of which is incorporated by reference in its
entirety.
[0326] Because sulfur is a strong and reactive reducing and
precipitating agent for most transition metals (see
Charerntanyarak, L., Heavy Metals Removal by Chemical Coagulation
and Precipitation. Water Science and Technology 1999, 39 (10-11),
135-138, which is incorporated by reference in its entirety),
sulfur was aim to be overproduced, whereas the wine industry has
attempted to inhibit sulfur production (FIG. 30). Overproducing
hydrogen sulfide serves as a powerful method for metal remediation
in the form of chemical precipitation. From here, either the first
or second bioremediation strategy can be employed to bind or
uptake, respectively, these sulfide-metal complexes.
[0327] To utilize this strategy for bioremediation, the sulfur
assimilatory cycle need to be interrupted at the point of sulfate
to sulfide conversion (HSO.sub.3.sup.-.fwdarw.S.sup.2- [STOP]) and
build up reactive sulfur in solution. Detection of hydrogen sulfide
can be indirectly monitored from released hydrogen sulfide gas.
Qualitative identification can be observed using lead acetate
strips while quantitative measurements can be performed using
sulfide detection columns, both of which undergo colorimetric
changes in the presence of sulfur (FIGS. 31A-31D). Monitoring color
changes shows a production rate of 2 ppm/hr with a maximum
production of 55 pm during a complete culture cycle (<24
hr).
[0328] The successful mutant, .DELTA.MET17, is able to produce
roughly 2 ppm/hr of hydrogen sulfide with a total of 55 ppm during
a complete inoculation experiment (>24 hr) in CSM. Cultures
seeded with cadmium or copper precipitate to CdS and CuS,
respectively, showed a conversion of 90.+-.5% of the initial metal
concentration (FIGS. 34A-34B). Bioprecipitated metal sulfides, such
as CdS, are regularly used as quantum dots. Reacted CdS from yeast
behaves with characteristic excitation and emission wavelengths of
that of typical quantum dots. The size of the CdS particles is
approximately 50 nm in diameter, consistent with the expected size
that contributes to the quantum confinement of electrons given the
distinct excitation and emission peaks (FIG. 32C).
[0329] These CdS particles are embedded in the cell wall using
cross-sectioning TEM (FIG. 33A). The cell wall can then be digested
using zymolase to release the particles, and a uniform distribution
of CdS particle sizes, as well as some CdS particles encapsulated
in biologically derived material (e.g., protein or cell wall
debris) were observed (FIG. 33B).
Packaging and Deployment
[0330] Another option is to store yeast in freeze-dried or active
dry packages, much as baker's yeast are stored for consumer use,
and distribute them for on-demand applications. Large quantities of
packaged yeast can be stored for later use during the events of
disaster spills or contamination leaks much like the BP oil spill,
Fukushima nuclear disaster, and the Flint water crisis in 2010,
2012, and 2014, respectively. Deployable units of the engineered
yeast can be created for on-site waste treatment. Options to create
such a device may include constructing a filtering device that
supports a resin-like bed of yeast.
[0331] Reducing the cost and scaling up the yeast technology can
take advantage of the already established infrastructure for
mass-producing yeast for consumer purposes. The beer, wine, and
pharmaceutical industries have optimized large-scale production of
yeast, so there is already developed infrastructure to produce
yeast in mass. The production and consumption of bread, beer, and
medicine can be concurrently used to clean contaminated waters
(FIGS. 34A-34B).
[0332] Other embodiments are within the scope of the following
claims.
Sequence CWU 1
1
9148DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotidemodified_base(1)..(1)a, c, t, g, unknown
or othermodified_base(3)..(5)a, c, t, g, unknown or
othermodified_base(7)..(7)a, c, t, g, unknown or
othermodified_base(9)..(11)a, c, t, g, unknown or
othermodified_base(13)..(13)a, c, t, g, unknown or
othermodified_base(15)..(17)a, c, t, g, unknown or
othermodified_base(19)..(19)a, c, t, g, unknown or
othermodified_base(21)..(23)a, c, t, g, unknown or
othermodified_base(25)..(25)a, c, t, g, unknown or
othermodified_base(27)..(29)a, c, t, g, unknown or
othermodified_base(31)..(31)a, c, t, g, unknown or
othermodified_base(33)..(35)a, c, t, g, unknown or
othermodified_base(37)..(37)a, c, t, g, unknown or
othermodified_base(39)..(41)a, c, t, g, unknown or
othermodified_base(43)..(43)a, c, t, g, unknown or
othermodified_base(45)..(47)a, c, t, g, unknown or other
1ndnnnkndnn nkndnnnknd nnnkndnnnk ndnnnkndnn nkndnnnk
48272DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotidemodified_base(1)..(1)a, c, t, g, unknown
or othermodified_base(3)..(5)a, c, t, g, unknown or
othermodified_base(7)..(7)a, c, t, g, unknown or
othermodified_base(9)..(11)a, c, t, g, unknown or
othermodified_base(13)..(13)a, c, t, g, unknown or
othermodified_base(15)..(17)a, c, t, g, unknown or
othermodified_base(19)..(19)a, c, t, g, unknown or
othermodified_base(21)..(23)a, c, t, g, unknown or
othermodified_base(25)..(25)a, c, t, g, unknown or
othermodified_base(27)..(29)a, c, t, g, unknown or
othermodified_base(31)..(31)a, c, t, g, unknown or
othermodified_base(33)..(35)a, c, t, g, unknown or
othermodified_base(37)..(37)a, c, t, g, unknown or
othermodified_base(39)..(41)a, c, t, g, unknown or
othermodified_base(43)..(43)a, c, t, g, unknown or
othermodified_base(45)..(47)a, c, t, g, unknown or
othermodified_base(49)..(49)a, c, t, g, unknown or
othermodified_base(51)..(53)a, c, t, g, unknown or
othermodified_base(55)..(55)a, c, t, g, unknown or
othermodified_base(57)..(59)a, c, t, g, unknown or
othermodified_base(61)..(61)a, c, t, g, unknown or
othermodified_base(63)..(65)a, c, t, g, unknown or
othermodified_base(67)..(67)a, c, t, g, unknown or
othermodified_base(69)..(71)a, c, t, g, unknown or other
2ndnnnkndnn nkndnnnknd nnnkndnnnk ndnnnkndnn nkndnnnknd nnnkndnnnk
60ndnnnkndnn nk 72396DNAArtificial SequenceDescription of
Artificial Sequence Synthetic
oligonucleotidemodified_base(1)..(1)a, c, t, g, unknown or
othermodified_base(3)..(5)a, c, t, g, unknown or
othermodified_base(7)..(7)a, c, t, g, unknown or
othermodified_base(9)..(11)a, c, t, g, unknown or
othermodified_base(13)..(13)a, c, t, g, unknown or
othermodified_base(15)..(17)a, c, t, g, unknown or
othermodified_base(19)..(19)a, c, t, g, unknown or
othermodified_base(21)..(23)a, c, t, g, unknown or
othermodified_base(25)..(25)a, c, t, g, unknown or
othermodified_base(27)..(29)a, c, t, g, unknown or
othermodified_base(31)..(31)a, c, t, g, unknown or
othermodified_base(33)..(35)a, c, t, g, unknown or
othermodified_base(37)..(37)a, c, t, g, unknown or
othermodified_base(39)..(41)a, c, t, g, unknown or
othermodified_base(43)..(43)a, c, t, g, unknown or
othermodified_base(45)..(47)a, c, t, g, unknown or
othermodified_base(49)..(49)a, c, t, g, unknown or
othermodified_base(51)..(53)a, c, t, g, unknown or
othermodified_base(55)..(55)a, c, t, g, unknown or
othermodified_base(57)..(59)a, c, t, g, unknown or
othermodified_base(61)..(61)a, c, t, g, unknown or
othermodified_base(63)..(65)a, c, t, g, unknown or
othermodified_base(67)..(67)a, c, t, g, unknown or
othermodified_base(69)..(71)a, c, t, g, unknown or
othermodified_base(73)..(73)a, c, t, g, unknown or
othermodified_base(75)..(77)a, c, t, g, unknown or
othermodified_base(79)..(79)a, c, t, g, unknown or
othermodified_base(81)..(83)a, c, t, g, unknown or
othermodified_base(85)..(85)a, c, t, g, unknown or
othermodified_base(87)..(89)a, c, t, g, unknown or
othermodified_base(91)..(91)a, c, t, g, unknown or
othermodified_base(93)..(95)a, c, t, g, unknown or other
3ndnnnkndnn nkndnnnknd nnnkndnnnk ndnnnkndnn nkndnnnknd nnnkndnnnk
60ndnnnkndnn nkndnnnknd nnnkndnnnk ndnnnk 96445PRTArabidopsis
thaliana 4Met Ala Asp Ser Asn Cys Gly Cys Gly Ser Ser Cys Lys Cys
Gly Asp1 5 10 15Ser Cys Ser Cys Glu Lys Asn Tyr Asn Lys Glu Cys Asp
Asn Cys Ser 20 25 30Cys Gly Ser Asn Cys Ser Cys Gly Ser Asn Cys Asn
Cys 35 40 45581PRTArabidopsis thaliana 5Met Ser Cys Cys Gly Gly Asn
Cys Gly Cys Gly Ser Gly Cys Lys Cys1 5 10 15Gly Asn Gly Cys Gly Gly
Cys Lys Met Tyr Pro Asp Leu Gly Phe Ser 20 25 30Gly Glu Thr Thr Thr
Thr Glu Thr Phe Val Leu Gly Val Ala Pro Ala 35 40 45Met Lys Asn Gln
Tyr Glu Ala Ser Gly Glu Ser Asn Asn Ala Glu Asn 50 55 60Asp Ala Cys
Lys Cys Gly Ser Asp Cys Lys Cys Asp Pro Cys Thr Cys65 70 75
80Lys669PRTArabidopsis thaliana 6Met Ser Ser Asn Cys Gly Ser Cys
Asp Cys Ala Asp Lys Thr Gln Cys1 5 10 15Val Lys Lys Gly Thr Ser Tyr
Thr Phe Asp Ile Val Glu Thr Gln Glu 20 25 30Ser Tyr Lys Glu Ala Met
Ile Met Asp Val Gly Ala Glu Glu Asn Asn 35 40 45Ala Asn Cys Lys Cys
Lys Cys Gly Ser Ser Cys Ser Cys Val Asn Cys 50 55 60Thr Cys Cys Pro
Asn65784PRTArabidopsis thaliana 7Met Ala Asp Thr Gly Lys Gly Ser
Ser Val Ala Gly Cys Asn Asp Ser1 5 10 15Cys Gly Cys Pro Ser Pro Cys
Pro Gly Gly Asn Ser Cys Arg Cys Arg 20 25 30Met Arg Glu Ala Ser Ala
Gly Asp Gln Gly His Met Val Cys Pro Cys 35 40 45Gly Glu His Cys Gly
Cys Asn Pro Cys Asn Cys Pro Lys Thr Gln Thr 50 55 60Gln Thr Ser Ala
Lys Gly Cys Thr Cys Gly Glu Gly Cys Thr Cys Ala65 70 75 80Ser Cys
Ala Thr862PRTArabidopsis thaliana 8Asn Ile Phe Ser Glu Leu Ile Asn
Phe Gln Asn Glu Gly His Glu Cys1 5 10 15Gln Cys Gln Cys Gly Ser Cys
Lys Asn Asn Glu Gln Cys Gln Lys Ser 20 25 30Cys Ser Cys Pro Thr Gly
Cys Asn Ser Asp Asp Lys Cys Pro Cys Gly 35 40 45Asn Lys Ser Glu Glu
Thr Lys Lys Ser Cys Cys Ser Gly Lys 50 55 6099PRTHuman influenza
virus 9Tyr Pro Tyr Asp Val Pro Asp Tyr Ala1 5
* * * * *
References